Flattop (fltp) is a novel biomarker for beta cell maturation

ABSTRACT

The present invention relates to the use of the biomarker Flattop (Fltp) for distinguishing mature β cells from immature progenitor β cells. The present invention further relates to a method for distinguishing a mature β cell from an immature progenitor β cell, the method comprising: determining the presence or absence of the biomarker Flattop (Fltp) in a β cell; wherein the presence of Fltp in the cell indicates that the cell is a mature β cell and wherein the absence of Fltp in the cell indicates that the cell is an immature progenitor β cell. Furthermore, the present invention relates to a method of identifying a compound suitable for differentiating immature progenitor β cells into mature β cells as well as to a method of identifying a compound suitable for preventing the de-differentiating of mature β cells.

The present invention relates to the use of the biomarker Flattop (Fltp)for distinguishing mature β cells from immature progenitor β cells. Thepresent invention further relates to a method for distinguishing amature β cell from an immature progenitor β cell, the method comprising:determining the presence or absence of the biomarker Flattop (Fltp) in aβ cell; wherein the presence of Fltp in the cell indicates that the cellis a mature β cell and wherein the absence of Fltp in the cell indicatesthat the cell is an immature progenitor β cell. Furthermore, the presentinvention relates to a method of identifying a compound suitable fordifferentiating immature progenitor β cells into mature β cells as wellas to a method of identifying a compound suitable for preventing thede-differentiating of mature β cells. The present invention additionallyrelates to a method of differentiating immature progenitor β cells intomature β cells as well as to a method of preventing de-differentiatingof mature β cells. In addition, the present invention also relates to akit for distinguishing mature β cells from immature progenitor β cellsand to a pharmaceutical composition for use in treating or preventingdiabetes.

In this specification, a number of documents including patentapplications and manufacturer's manuals are cited. The disclosure ofthese documents, while not considered relevant for the patentability ofthis invention, is herewith incorporated by reference in its entirety.More specifically, all referenced documents are incorporated byreference to the same extent as if each individual document wasspecifically and individually indicated to be incorporated by reference.

The islet of Langerhans is a complex micro-organ that contains fiveendocrine cell types (α, β, δ, PP, and ε cells), arterial and venousblood supply as well as sympathetic, parasympathetic and sensory neuroninnervation (In't Veld and Marichal 2010). The sole function of thismicro-organ is to regulate energy metabolism. To fulfill this function,glucose-sensitive β cells are organized around vessels in rosette-likestructures to secrete at the apical PM insulin into the blood streamupon food intake (Bonner-Weir 1988). β cells are functionally coupled bygap junctions and establish cell-matrix adhesion to blood vessels, whichis required for optimal β cell function, suggesting that cell polaritymight also be important (Eberhard and Lammert 2009). Some support forthis idea comes from the β cell-specific knock out of the Lkb1 kinasethat is involved in polarity establishment and mTOR regulation (Granot,Swisa et al. 2009). This leads to a dramatic increase in insulinsecretion as well as cell polarity and cilia positioning defects.However, due to the dual role of Lkb1 in polarity establishment andenergy control it is difficult to conclude on the requirement of cellpolarity for β cell function.

As mentioned above, β cells are arranged around blood vessels and areoften, but not always, in direct contact to blood vessels on the apicaland basal side (Bonner-Weir 1988). It is uncertain if this heterogeneityin tissue organization also reflects heterogeneity in β cell function.Salomon and Meda already reported that β cells in vitro areheterogeneous in their insulin secretion capacity depending on cell-cellcoupling (Salomon and Meda 1986). One year later these results wereconfirmed in vivo and it was shown that central versus peripheral, butalso splenic versus duodenal β cells showed different insulin secretioncapacities upon glucose challenge (Stefan, Meda et al. 1987). Furtherevidence for the heterogeneity of β cells was provided by thedemonstration that β cells differ in their glucose-responsiveness due tothe higher expression and activity of glucokinase, rather thandifferences in Glut2 mRNA or glucose transport (Heimberg, De Vos et al.1993). In contrast, a pancreatic multipotent progenitor (PMP) populationwas described in mouse and human that resides in the islet, expressesinsulin and low levels of Glut2 and shows extensive proliferativecapacity, self-renewal and multipotency (Smukler, Arntfield et al.2011). Recently, the expression of a fluorescent marker under the mouseinsulin promoter allowed to distinguish subpopulations of β cells thatdiffer in their granularity, size and secretion capacity, but the reasonfor these differences is still under debate (Katsuta, Aguayo-Mazzucatoet al. 2012). Thus although β cell heterogeneity was discovered over 25years ago, the underlying principal of this phenomenon is stillcontroversially discussed.

During early postnatal islet neogenesis in mouse, naïve β cells arestill organized in solitary or cord-like structures and acquire thetypical spheric 3D islet architecture in the first two weeks afterbirth. These naïve β cells secrete insulin at low thresholds and do notreach maturation until a couple weeks after birth when they start toexpress the currently only known maturation marker, Urocortin 3 (Ucn3)(Blum, Hrvatin et al. 2012). Functional maturation of β cells derivedfrom progenitor cells is currently one of the major hurdles forcell-replacement therapy (Pagliuca and Melton 2013). Moreover, it wasrecently proposed that β cell de-differentiation is one of themechanisms of β cell failure in type 2 diabetes (Talchai, Xuan et al.2012). Further support for this idea comes from a recent report thatPdx1 maintains β cell identity and β cell specific knock-out of Pdx1causes an β- to α-cell reprogramming (Gao, McKenna et al. 2014).

Thus, despite the fact that a lot of effort has been invested intoinvestigating β cell maturation, there is still a need to identify novelmaturation markers in order to allow understanding the pathomechanismsunderlying diabetes, enable regeneration of de-differentiated β cells inislets of Langerhans, and generate functional mature β cells frompluripotent stem cells for cell replacement therapy.

This need is addressed by the provision of the embodiments characterizedin the claims.

Accordingly, the present invention relates in a first aspect to the useof the biomarker Flattop (Fltp) for distinguishing mature β cells fromimmature progenitor β cells.

The term “biomarker” according to the present invention relates to therecited marker in any of its naturally occurring forms, includingnucleic acid molecules such as e.g. DNA, including cDNA or genomic DNA,and RNA as well as proteins.

As used herein, the term “Flattop (Fltp)” refers to a recentlyidentified gene expressed in the embryonic node, but also active inother monociliated tissues such as the sensory organs of the inner ear(IE), duct and islets of the pancreas as well as in testis (Lange, Gegget al. 2012). Additionally, Fltp mRNA is expressed in multiciliatedepithelial cells of the lung and of the choroid plexi in the brain.Loss-of-gene function in multiciliated lung epithelial cells has beenshown to lead to basal body docking defects, which result in loss ofcilia. In addition, loss of Fltp causes planar cell polarity (PCP)phenotypes in the IE and the lung. Further, Fltp localizes in a PCP-likeasymmetric fashion in sensory IE cells and genetically interacts withthe core PCP molecule Celsr1.

Human Flattop (Fltp) is, for example, represented by the Entrez Gene ID257177 and UniProt ID UPF0740 protein C1Orf192, as shown in SEQ ID NOs:1 and 2. Mouse Flattop is, for example, represented by the Entrez GeneID 75472 and UniProt ID Q6P8X9, as shown in SEQ ID NOs: 3 and 4.

β cells are unique cells present in the pancreas, in particular in theendocrine structures thereof, i.e. the islets of Langerhans. β cells areone of at least five different types of islet cells that produce andsecrete hormones directly into the bloodstream and they produce, storeand release the hormone insulin. Usually, β cells are perfect sensors ofblood glucose levels and secrete just the right amount of insulin intothe bloodstream to systemically regulate glucose and energy homeostasis(Lickert 2013). Diabetes, a disease that affects a large number ofpeople worldwide, is associated with β cell failure. Type 1 diabetesresults from autoimmune destruction of β cells, whereas in type 2diabetes a failure of β cells to compensate for peripheral insulinresistance leads to exhaustion, dedifferentiation, and loss of functionβ cell mass. Preferably, the cells referred to herein are from amammalian animal, such as e.g. a rodent, preferably a mouse, a rat or arabbit; a pig, a dog, a cat or a primate. More preferably, the cells arehuman or mouse cells.

The term “mature β cells”, as used herein, refers to highly polarized βcells that secrete insulin in response to a glucose stimulus. Thisrequires molecular machineries that measure blood glucose and produce,process and secrete insulin. Mature β cells show almost no proliferationand are clustered around blood vessels, towards which they secrete theinsulin (Eberhard and Lammert 2009). Mature β cells are furthercharacterized by the presence of the maturation marker Ucn3 (Blum,Hrvatin et al. 2012). In addition, as shown in Example 6 below, mature βcells were found herein to show a significant enrichment of genes thatare important for mature β cell function, such as genes involved inmetabolic processes, glucose metabolism, mitochondria and insulinsecretion. Anatomically, mature β cells cluster around blood vessels inrosette-like structures and show apical-basal polarity with insulingranules docking towards the apical lumen (Bonner-Weir 1988).

In accordance with the present invention, the terms “immature progenitorβ cells” and “naïve progenitor β cells”, which are used interchangeablyherein, refer to highly proliferative and low polarized progenitor cellsthat produce and secrete only low amounts of insulin and express lowlevels of the glucose transporter Glut2 (Smukler, Arntfield et al.2011). Immature β cells are likely in loose contact with each other andmight not form clusters around blood vessels, contrary to mature βcells. Known markers for all β cells, including immature progenitor βcells, but also mature β cells are Pdx1, Nkx6.1 and insulin. However, incontrast to mature β cells, immature progenitor cells lack expression ofFlattop and express genes like Glp1r, Gck, Insrr, Slc2a2 to an at least2-fold lower extent as compared to mature β cells, but show an at least2-fold higher expression in Smarca1 and Sstr2.

In addition, as shown in Example 6 below, immature progenitor β cellswere found herein to show a significant enrichment of further genes thatassociate with cell proliferation, actin binding, Wnt/PCP, TGFreceptor-, G-protein coupled receptor- and ERK-signaling transduction.Anatomically, and in contrast to mature β cells, immature progenitor βcells do not form rosette-like structures but appear to be lying next tothe rosettes. Moreover, it appears that immature progenitor β cells donot show the apical-basal polarity with insulin granules docking towardsthe apical lumen, found in mature β cells (Bonner-Weir 1988).

As is shown in the appended examples, immature β cells lack expressionof Fltp while mature β cells express this biomarker. Accordingly, thepresence of Fltp can be used to distinguishing mature β cells fromimmature progenitor β cells. To this end, the presence or absence ofFltp in β cells of interest can be determined, wherein the presence ofFltp indicates that the β cell is a mature β cell and wherein theabsence of Fltp indicates that the β cell is an immature progenitor βcell.

As used herein, the term “determining the [ . . . ] presence or absence”refers to the analysis whether the recited molecule is present or absentin cells comprised in the sample investigated. The molecule isconsidered present in accordance with the present invention when it isdetected in amounts exceeding the standard procedural error, such as forexample observed in the form of background staining obtained inimmunohistochemical or western blot analyses. Such procedural errors canbe determined according to established procedures, for example byanalyzing non-disease control samples or by omitting certain steps orcompounds in the procedure, such as for example a primary antibody inimmunohistochemical stainings or a template in nucleic acidamplification techniques etc. In the case that the amount of moleculedetected corresponds to or is less than the standard procedural error,e.g. the background staining in an immunohistochemical analysis, themolecule is considered as being absent in the sample.

The use of Fltp fusion proteins comprising a detectable moiety or theuse of Fltp reporter proteins for determining the presence or absence ofFltp is also encompassed by the present invention.

In the first case, expression of Fltp leads to the concomitantexpression of the detectable moiety, for example a tag such as aHis-tag, FLAG-tag, TAP-tag or myc-tag; a luminescent or fluorescentmarker such as e.g. luciferase, in particular bacterial luciferase(luxAB), Photinus luciferase, Renilla luciferase; fluorescent proteinssuch as e.g. green fluorescent protein (GFP) including enhanced GFP(EGFP), yellow fluorescent protein (YFP), including in particular theimproved Venus fluorescent protein, red fluorescent protein (RFP), cyanfluorescent protein (CFP), coral-derived photoproteins including DSRed,HcRed, AmCyan, ZsGreen, ZsYellow, AsRed; or an enzymatic marker, such ase.g. β-galactosidase, CAT, β-glucuronidase, β-xylosidase, XylE (catecholdioxygenase), TreA (trehalase), alkaline phosphatase or secretedalkaline phosphatase.

As is shown in the appended examples, a Flattop-Venus fusion protein wasgenerated by directly fusing the Venus fluorescent protein to theopen-reading frame of Flattop. Mice expressing this Flattop-Venus fusionprotein in all tissues were generated, wherein the Flattop-Venus fusionprotein is expressed in equal amounts to the wild-type Flattop protein.This Flattop-Venus fusion reporter has the advantage that it isexpressed in physiological amounts, shows normal protein turnover andnormal subcellular localization.

In the latter case, the expression of a detectable reporter protein isunder the control of the Fltp promoter and, preferably, the naturallyoccurring regulatory sequences of Fltp expression. Such a reporterprotein can be expressed either instead of Fltp expression, or inaddition to Fltp expression, and can be detected by the methodsdescribed below. The detectable reporter protein may e.g. be any of theluminescent, fluorescent or enzymatic markers recited above. As alsodescribed in the examples below, a Fltp^(ZV) knock-in/knock-out allelewas generated where the entire ORF was replaced by a multicistroniclacZ-Venus reporter cassette that contained, amongst others, a brightHistone 2B (H2B)-Venus fluorescent reporter gene and allows to exploreFltp expression and function in vivo.

In accordance with the present invention, the presence or absence of aspecific molecule, such as e.g. Fltp, a reporter protein, or a Fltpfusion protein, can be determined either on (i) the protein level, (ii)the nucleic acid level, or (iii) a combination thereof.

Methods for the determination of the presence or absence of a proteininclude but are not limited to methods such as e.g. immunohistochemicalmethods, immunocytochemical methods, live cell imaging including,without being limiting high-content screening via live reporters (suchas e.g. Opereta, Operon), bulk fluorescent measurements (e.g. Envision),quantitative fluorescence imaging/quantitative fluorescence microscopy,ELISA, FACS analysis or Cytof® Mass Cytometry as well as immunoblotting,such as e.g. Western blotting, or polyacrylamide gel electrophoresis inconjunction with protein staining techniques such as Coomassie Brilliantblue or silver-staining. Preferably, screening for Flattop protein iscarried out by immuohistochemistry or immunocytochemistry usingantibodies or alternative binding molecules specific for the Flattopprotein or by live cell imaging using e.g. a reporter system.

The term “antibody”, as used in accordance with the present invention,comprises polyclonal and monoclonal antibodies, as well as derivativesor fragments thereof, which still retain their binding specificity.Antibody fragments or derivatives comprise, inter alia, Fab or Fab′fragments as well as Fd, F(ab′)₂, Fv or scFv fragments; see, for exampleHarlow and Lane “Antibodies, A Laboratory Manual”, Cold Spring HarborLaboratory Press, 1988 and Harlow and Lane “Using Antibodies: ALaboratory Manual” Cold Spring Harbor Laboratory Press, 1999. The term“antibody” also includes embodiments such as chimeric (human constantdomain, non-human variable domain), single chain and humanized (humanantibody with the exception of non-human CDRs) antibodies.

Various techniques for the production of antibodies are well known inthe art and described, e.g. in Harlow and Lane (1988) and (1999). Forexample, the antibodies can be produced as peptidomimetics. Further,techniques described for the production of single chain antibodies (see,inter alia, U.S. Pat. No. 4,946,778) can be adapted to produce singlechain antibodies specific for their respective target. Also, transgenicanimals or plants (see, e.g., U.S. Pat. No. 6,080,560) may be used toexpress (humanized) antibodies specific for the target of thisinvention. Most preferably, the antibody is a monoclonal antibody, suchas a human or humanized antibody. For the preparation of monoclonalantibodies, any technique which provides antibodies produced bycontinuous cell line cultures can be used. Examples for such techniquesare described, e.g. in Harlow and Lane (1988) and (1999) and include thehybridoma technique originally developed by Köhler and Milstein (Kohlerand Milstein 1975), the trioma technique, the human B-cell hybridomatechnique (Kozbor 1983) and the EBV-hybridoma technique to produce humanmonoclonal antibodies (Cole 1985). Surface plasmon resonance as employedin the BIAcore system can be used to increase the efficiency of phageantibodies which bind to a target protein (Schier and Marks 1996). It isalso envisaged in the context of this invention that the term “antibody”comprises antibody constructs which may be expressed in cells, e.g.antibody constructs which may be transfected and/or transduced via,inter alia, viruses or plasmid vectors.

As is shown in the appended examples, two different polyclonal rabbitantibodies were raised against a central and C-terminal epitope. Theseantibodies can be used as primary antibodies to detect the protein intissues or cell cultures and using secondary antibodies eitherconjugated to horseradish peroxidase, alkaline phosphatase orfluorescent dyes. Based on these epitopes shown in examples below, it ispossible without further ado to prepare antibodies, for exampleemploying any of the methods known in the art referred to above.

Alternative binding molecules include, without being limiting, proteinsor peptides in which specific binding properties have been introduced,for example by mutagenesis, into a protein scaffold with suitablebiophysical properties. Ideally, small globular proteins that are easyto express and purify, which are soluble and stable, which do notaggregate and which are non-immunogenic, are used as a scaffold. So farmore than 50 proteins of this class have been described, among themaffibodies (which are based on the Z-domain of staphylococcal protein A(Feldwisch and Tolmachev 2012)), adnectins (based on the tenth domain ofhuman fibronectin (Gebauer and Skerra 2009)), anticalins (derived fromlipocalins (Beste, Schmidt et al. 1999, Gebauer and Skerra 2009)),DARPins (derived from ankyrin repeat proteins (Gebauer and Skerra2009)), avimers (based e.g. on multimerised Low Density LipoproteinReceptor (LDLR)-A (Weidle, Auer et al. 2013)), nanofitins (derived fromthe DNA binding protein Sac7d of Sulfolobus acidocaldarius (Mouratou,Behar et al. 2012)), affilins (structurally derived from gamma-Bcrystalline or Ubiquitin (Weidle, Auer et al. 2013)), Kunitz domainpeptides (derived from the Kunitz domains of various protease inhibitors(Weidle, Auer et al. 2013)) and Fynomers®, which are derived from thehuman Fyn SH3 domain (Bertschinger, Grabulovski et al. 2007,Grabulovski, Kaspar et al. 2007, Gebauer and Skerra 2009, Schlatter,Brack et al. 2012). These proteins or peptides are well known in theart.

As mentioned herein above, and depending on the detection methodemployed, the determination of Fltp expression can be employed by liveimaging, i.e. methods where cells are or tissue is kept alive duringobservation, or on fixed samples, i.e. where cells or tissues areimmobilized and do not survive the imaging process.

The relative amount of the protein of interest is often determined bycomparing the total measured amount of protein, e.g. in form of thefluorescence intensity of a labeled protein, derived from a sample ofinterest with the total measured amount of protein obtained from acontrol sample. Also of use in protein quantification is the AgilentBioanalyzer technique.

Determination on the nucleic acid level refers to the determination ofthe presence or absence of a nucleic acid molecule encoding the proteinof interest and that is only present (i.e. up-regulated) when expressionof said protein has been activated. Preferably, said nucleic acidmolecule is mRNA or a cDNA obtained from mRNA. It will be appreciatedthat genomic DNA is excluded in accordance with the present invention.

Methods for determining presence or absence of a molecule on the nucleicacid level include, but are not limited to hybridization assays, nucleicacid amplification assays or sequencing assays.

Examples for hybridization assays comprise, without limitation, in situRNA hybridization, Northern and Southern blot assays. These methods arewell known in the art and have been described, e.g. in Michael Green andJoseph Sambrook Molecular Cloning: A Laboratory Manual (Fourth Edition).

Non-limiting examples for nucleic acid amplification assays and means toperform such include PCR, (including nested PCR, RT-PCR, quantitative(real-time) detection, PCR extension assays, nucleic acid sequence baseamplification (NASBA), single-strand confirmation polymorphism (SSCP)PCR, PCR-restriction enzyme fragment length polymorphism (RFLP)analysis), amplification refractory mutation systems (ARMSTM) andamplification refractory mutation system linear extension (ALEXTM)assays. Details of such methods can be found in art, (Newton, Graham etal. 1989, Haque, Hehir et al. 1998, Pissard, Huynh et al. 2002, Kakavas,Noulas et al. 2006, Steemers, Chang et al. 2006). Quantitative PCR canfor example be used to measure Fltp levels in islet cells. To this end,cDNA can first be linear amplified and the amplified cDNA can then besubjected to quantitative PCR.

Examples for sequencing assays comprise without limitation approaches ofsequence analysis by direct sequencing, fluorescent SSCP in an automatedDNA sequencer and Pyrosequencing. These procedures are common in theart, see e.g. Adams “Automated DNA Sequencing and Analysis” and Alphey,“DNA Sequencing: From Experimental Methods to Bioinformatics” (Ramon,Braden et al. 2003, Meng, Hager et al. 2005). For example, RNAsequencing can be used to analyze mRNA levels in Flattop positive andnegative FACS-isolated islet cell populations.

It is particularly preferred in accordance with the embodiments of thepresent invention that the presence or absence of Fltp is determined byimmunohistochemistry, RNA in situ hybridisation, quantitative PCR or acombination thereof. Preferred antibodies for use in determining thepresence or absence (or the amount) of Fltp are the antibodies describedin the Examples below, as well as any antibodies directed to the epitopeshown in FIG. 11, i.e. epitopel: DNPDEPQSSHPSAGHT (mouse; SEQ ID NO:5);NSPDELQSSHPSAGHT (human; SEQ ID NO:6) and mP17Rik-116-135:KPFDPDSQTKQKKSVTKTVQ (mouse; SEQ ID NO:7); and the corresponding humanepitope: KPHDPDSQKKLRKKSITKTVQ (human; SEQ ID NO:8).

Preferred primers for use in determining the presence or absence (or theamount) of Fltp are the primers described in the Examples below, inparticular the following primers: human forward primer5′-ACCTGGCAAATGCCTCTGAA-3′ (SEQ ID NO:9); human reverse primer5′-GGATCATGGGGCTTGCCTAA-3′ (SEQ ID NO:10) and human forward primer5′-CCTGACCTCCCGTACAACTG-3′ (SEQ ID NO:11); human reverse primer5′-TGGATCATGGGGCTTGCCTA-3′ (SEQ ID NO:12); mouse:5′-AGCCATACCACATTTGTAGAGG-3′ (SEQ ID NO:13); 5′-CAGCATGGCATAGATCTGGAC-3(SEQ ID NO:14)′; 5′-GAGGCTGACTGGGAACAATC-3′ (SEQ ID NO:15).

Preferred probes for use in determining the presence or absence (or theamount) of Fltp is for example the probe shown in SEQ ID NO: 16, whichcan be prepared using e.g. the primers shown in SEQ ID NOs: 17 and 18.

In accordance with the present invention, it was shown that duringpost-natal islet neogenesis β cells gradually receive increased Wnt/PCPsignaling measured by a Fltp reporter gene, which correlates with 3Dislet formation and β cell maturation. Flattop activity rises in α-,PP-, δ-, and ε-cells up to 45% and in β cells up to 70% in the first twoweeks of development. In adult islets, the Fltp-negative β cells show aroughly 2-fold higher proliferation rate when compared to theFltp-positive β cells, which strongly increases to a four-fold increaseupon pregnancy. Fltp loss-of-function was found to lead to 1^(st) phaseinsulin secretion defects and shows that PCP-mediated cytoskeletalrearrangements, basal body positioning and likely primary cilia functionis required for mature beta cell function. This is further supported bythe identification of SNPs in the human FLTP gene, which associate withinsulin secretion defects.

The isolation of Fltp reporter negative and Fltp reporter expressing βcells from knock-in reporter animals allowed for the first time toanalyze the differential molecular properties of these naïve and matureβ cell populations. Strikingly, the expression of maturation andpolarity markers, glycolysis enzyme and signaling receptors is markedlydifferent between these β cell populations. This opens new possibilitiesfor a pharmacological targeting of the proliferative vs. maturefunctions of β cells.

In addition, it was shown by genetic lineage tracing that the highlyproliferative Fltp-negative progenitor cells can convert intoFltp-positive, mature β cells. Together, these findings identify Fltp asa novel marker and PCP as the maturation principal of β cells, whichsolves a long-standing mystery of β cell heterogeneity and opens newavenues for β cell regeneration.

Moreover, these results hints towards the existence of a pancreaticmultipotent progenitor (PMP) population that has high self-renewingcapacity, can differentiate in all endocrine lineages and resides inmouse and human adult islets of Langerhans (Smukler, Arntfield et al.2011). The identification of heterogeneous β cell populations that haveeither increased proliferative capacity or a more mature phenotype opensnew possibilities of triggering these specific β cell features in thefuture. For example, the identification of differentially expressedsignaling receptors and pathways can form a pharmacological entry pointfor regenerative therapies.

Additionally, the identification of Fltp as a β cell maturation markerwill also improve the generation of functional mature β cells frompluripotent stem cells. At present, only naïve, immature andpoly-hormonal insulin-producing cells can be produced in vitro. Theidentification of the PCP effector gene Fltp indicates that 3Denvironment is important for functional β cell maturation. Moreover, theFltp PCP reporter gene allows screening for signals, miRNAs and smallmolecules that activate PCP signaling and lead to functional β cellmaturation. In addition, in light of recent findings that β cellde-differentiation contributes to β cell failure in type 2 diabetes, theidentification of Fltp as a novel maturation marker allows a moredetailed study of the pathogenesis of type 2 diabetes in mouse and man.

Overall, the findings of the present invention resolve the long-standingmystery of β cell heterogeneity and strongly suggest that tissuepolarity influences the heterogeneous polarity status of endocrinecells. In terms of β cells, this difference in polarity status directlyassociates with different biological function. The discovery of thisprinciple and Fltp as a PCP reporter allows deciphering pathomechanismsof diabetes and enables for novel approaches for regenerative therapy.

The present invention further relates to a method for distinguishing amature β cell from an immature progenitor β cell, the method comprising:determining the presence or absence of the biomarker Flattop (Fltp) in aβ cell; wherein the presence of the Fltp in the cell indicates that thecell is a mature β cell and wherein the absence of Fltp in the cellindicates that the cell is an immature progenitor β cell.

In accordance with the present invention, the method for distinguishinga mature β cell from an immature progenitor β cell is not limited to asingle cell. Instead, also a plurality of mature β cells can bedistinguished from a plurality of immature progenitor β cells.

The term “comprising”, as used herein, denotes that further steps and/orcomponents can be included in addition to the specifically recited stepsand/or components. However, this term also encompasses that the claimedsubject-matter consists of exactly the recited steps and/or components.

The definition of the term “determining the [ . . . ] presence orabsence” recited above as well as the above described preferred methodsof achieving such a determination apply mutatis mutandis also to thismethod of the invention.

In a preferred embodiment of this method of the invention, the presenceor absence of Fltp is determined by immunohistochemistry, quantitativePCR, FACS sorting or a combination thereof. Preferred antibodies,primers and probes have been described in detail above.

The present invention further relates to a method of identifying acompound suitable for differentiating immature progenitor β cells intomature β cells, the method comprising: (a) contacting a cell populationcomprising immature progenitor β cells with a test compound; and (b)subsequently determining the presence or expression level of thebiomarker Flattop (Fltp) in the β cells comprised in the cellpopulation; wherein the presence of Fltp, or an increased expressionlevel of Fltp, in the β cells comprised in the cell population after thecontacting with the test compound is indicative of a compound suitablefor differentiating immature progenitor β cells into mature β cells.

This embodiment relates to a screening assay for the identification ofcompounds capable of differentiating β cells, wherein Fltp is used as abiomarker to detect said differentiation. All definitions and preferredembodiments provided herein with regard to the use of the biomarkerFlattop (Fltp) and the method for distinguishing mature from immature βcells apply mutatis mutandis also to this screening method of theinvention.

As used herein, the term “differentiating β cells” refers to convertingimmature, naïve progenitor β cells to mature β cells.

Essentially any compound can be assayed in accordance with the methodsof the present invention. Such compounds include organic or inorganicmolecules. Organic molecules relate or belong to the class of chemicalcompounds having a carbon basis, the carbon atoms linked together bycarbon-carbon bonds, including biological entities such as e.g.proteins, sugars, nucleic acids, lipids. The original definition of theterm organic related to the source of chemical compounds, with organiccompounds being those carbon-containing compounds obtained from plant oranimal or microbial sources. Organic compounds can be natural orsynthetic. Small organic molecules preferably have a molecular weight ofabout 500 Da or below. Inorganic compounds are derived from mineralsources and include all compounds without carbon atoms (except carbondioxide, carbon monoxide and carbonates). There are many suppliers ofsuch compounds, including Sigma (St. Louis, Mo.), Aldrich (St. Louis,Mo.), Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-Biochemica Analytika(Buchs, Switzerland), for example. In addition, compounds to be analysedmay be synthesized by methods known in the art. Test compounds may becomprised in compound libraries of diverse or structurally similarcompounds (e.g., combinatorial chemistry synthesized libraries) and aplurality of test compounds in a library can be assayed simultaneously.Optionally, test compounds derived from different libraries can bepooled for simultaneous evaluation. A library can comprise a randomcollection of molecules. Alternatively, a library can comprise acollection of molecules having a bias for a particular sequence,structure, or conformation. Methods for preparing libraries containingdiverse populations of various types of molecules are known in the art(Brenk, Schipani et al. 2008, Quinn, Carroll et al. 2008). Numerouslibraries are also commercially available.

Preferred compounds for screening for their potential in differentiatingβ cells include, without being limiting, small molecules, antisensenucleic acid molecules, siRNA, shRNA, miRNA, antibodies, aptamers orribozymes as well as hormones and cytokines. Such compounds may beparticularly suitable as inhibitors of pathways that are involved in themaintenance of an undifferentiated state of β cells, e.g. by blockingspecific binding sites of a relevant target molecule(s) within saidpathway. Alternatively, or additionally, such compounds may also act asactivators of pathways that drive the differentiation of β cells, suchas e.g. the Wnt/PCP pathway, as described in more detail below.

A “small molecule” according to the present invention may be, forexample, an organic or inorganic molecule, as defined herein above.Preferably, the small molecule has a molecular weight of less than about2000 amu, or less than about 1000 amu, such as less than about 500 amu,and even more preferably less than about 250 amu. The size of a smallmolecule can be determined by methods well-known in the art, e.g., massspectrometry. The small molecules may be designed, for example, based onthe crystal structure of the target molecule, where sites presumablyresponsible for the biological activity, can be identified and verifiedin in vivo assays such as in vivo high-throughput screening (HTS)assays.

The term “antisense nucleic acid molecule”, as used herein, is known inthe art and refers to a nucleic acid which is complementary to a targetnucleic acid, i.e. a nucleic acid encoding the target protein. Anantisense molecule in accordance with the invention is capable ofinteracting with the target nucleic acid; more specifically it iscapable of hybridizing with the target nucleic acid. Due to theformation of the hybrid, transcription of the target gene(s) and/ortranslation of the target mRNA is reduced or blocked. Standard methodsrelating to antisense technology have been described (Melani, Rivoltiniet al. 1991).

As used herein, the term “siRNA”, refers to “small interfering RNA”,also known as “short interfering RNA” or “silencing RNA”, and relates toa class of 18 to 30, preferably 19 to 25, most preferred 21 to 23 oreven more preferably 21 nucleotide-long double-stranded RNA moleculesthat play a variety of roles in biology. siRNAs naturally found innature have a well defined structure: a short double-strand of RNA(dsRNA) with 2-nt 3′ overhangs on either end. Each strand has a 5′phosphate group and a 3′ hydroxyl (—OH) group. The double-stranded RNAmolecule or a metabolic processing product thereof is capable ofmediating target-specific nucleic acid modifications, particularly RNAinterference, where the siRNA interferes with the expression of aspecific gene, and/or DNA methylation.

siRNAs can be exogenously (artificially) introduced into cells to bringabout the specific knockdown of essentially any gene of interest ofwhich the sequence is known. Exogenously introduced siRNAs may be devoidof overhangs at their 3′ and 5′ ends, however, it is preferred that atleast one RNA strand has a 5′- and/or 3′-overhang. In general, any RNAmolecule suitable to act as siRNA is envisioned in the presentinvention. The most efficient silencing was so far obtained with siRNAduplexes composed of 21-nt sense and 21-nt antisense strands, paired ina manner to have 2-nt 3′ overhangs on either end. 2′-deoxynucleotides inthe 3′ overhangs are as efficient as ribonucleotides, but are oftencheaper to synthesize and probably more nuclease resistant.

The term “shRNA”, as used herein, refers to “short hairpin RNA” andrelates to a sequence of RNA that makes a tight hairpin turn thattypically can be used to silence gene expression via RNA interference.shRNA can for example use a vector introduced into cells, in which casepreferably the U6 promoter is utilized to ensure that the shRNA isalways expressed. This vector is usually passed on to daughter cells,allowing the gene silencing to be inherited. The shRNA hairpin structureis cleaved by the cellular machinery into siRNA, which is then bound tothe RNA-induced silencing complex (RISC). This complex binds to andcleaves mRNAs which match the siRNA that is bound to it.

Preferably, si/shRNAs to be used in the present invention are chemicallysynthesized using conventional methods that, for example, appropriatelyprotected ribonucleoside phosphoramidites and a conventional DNA/RNAsynthesizer. Non-limiting examples of suppliers of RNA synthesisreagents are Proligo (Hamburg, Germany), Dharmacon Research (Lafayette,Colo., USA), Pierce Chemical (part of Perbio Science, Rockford, Ill.,USA), Glen Research (Sterling, Va., USA), ChemGenes (Ashland, Mass.,USA), and Cruachem (Glasgow, UK). Most conveniently, siRNAs or shRNAsare obtained from commercial RNA oligo synthesis suppliers, which sellRNA-synthesis products of different quality and costs. In general, theRNAs applicable in the present invention are conventionally synthesizedand are readily provided in a quality suitable for RNAi.

The term “miRNA”, as used herein, refers to “microRNAs” and relates tosingle-stranded RNA molecules which, as endogenous RNA molecules,regulate gene expression. Binding to a complementary mRNA transcripttriggers the degradation of said mRNA transcript through a processsimilar to RNA interference. Accordingly, miRNA may be employed as aninhibitor of signaling pathways in accordance with the presentinvention.

The term “antibody”, as used in accordance with the present invention,has been defined herein above.

An “aptamer” is a DNA or RNA molecule that binds other molecules, suchas nucleic acids, proteins, small organic compounds, and even entireorganisms. A database of aptamers is maintained in the world wide web ataptamer.icmb.utexas.edu/.

More specifically, aptamers can be classified as nucleic acid aptamers(i.e. DNA or RNA aptamers) or peptide aptamers. Nucleic acid aptamersconsist of (usually short) strands of oligonucleotides and have beenengineered through repeated rounds of in-vitro selection orequivalently, SELEX (systematic evolution of ligands by exponentialenrichment) to bind to various molecular targets such as smallmolecules, proteins, nucleic acids, and even cells, tissues andorganisms. Peptide aptamers are proteins that are designed to interferewith other protein interactions inside cells. They consist of a variablepeptide loop attached at both ends to a protein scaffold. This doublestructural constraint greatly increases the binding affinity of thepeptide aptamer to levels comparable to an antibody's (nanomolar range).The variable loop is typically comprised of 10 to 20 amino acids, andthe scaffold may be any protein which has good solubility properties.Currently, the bacterial protein Thioredoxin-A is the most used scaffoldprotein, the variable loop being inserted within the reducing activesite, which is a -Cys-Gly-Pro-Cys-loop in the wild-type protein, the twocysteine lateral chains being able to form a disulfide bridge. Peptideaptamer selection can be made using different systems, but the most usedis currently the yeast two-hybrid system.

In addition to their discriminate recognition, aptamers offer advantagesover antibodies as they can be engineered completely in a test tube, arereadily produced by chemical synthesis, possess desirable storageproperties, and elicit little or no immunogenicity in therapeuticapplications. Non-modified aptamers are cleared rapidly from thebloodstream, with a half-life of minutes to hours, mainly due tonuclease degradation and clearance from the body by the kidneys, aresult of the inherently low molecular weight of aptamers. Severalmodifications, such as 2′-fluorine-substituted pyrimidines, polyethyleneglycol (PEG) linkage, fusion to albumin or other half-life extendingproteins etc. are available to scientists with which the half-life ofaptamers easily can be increased to the day or even week time scale.

A ribozyme (from ribonucleic acid enzyme, also called RNA enzyme orcatalytic RNA) is an RNA molecule that catalyzes a chemical reaction.Many natural ribozymes catalyze either their own cleavage or thecleavage of other RNAs, but they have also been found to catalyze theaminotransferase activity of the ribosome. Non-limiting examples ofwell-characterized small self-cleaving RNAs are the hammerhead, hairpin,hepatitis delta virus, and in vitro-selected lead-dependent ribozymes,whereas the group I intron is an example for larger ribozymes. Theprinciple of catalytic self-cleavage has become well established in thelast 10 years. The hammerhead ribozymes are characterized best among theRNA molecules with ribozyme activity. Since it was shown that hammerheadstructures can be integrated into heterologous RNA sequences and thatribozyme activity can thereby be transferred to these molecules, itappears that catalytic antisense sequences for almost any targetsequence can be created, provided the target sequence contains apotential matching cleavage site. The basic principle of constructinghammerhead ribozymes is as follows: An interesting region of the RNA,which contains the GUC (or CUC) triplet, is selected. Twooligonucleotide strands, each usually with 6 to 8 nucleotides, are takenand the catalytic hammerhead sequence is inserted between them.Molecules of this type were synthesized for numerous target sequences.They showed catalytic activity in vitro and in some cases also in vivo.The best results are usually obtained with short ribozymes and targetsequences.

A recent development, also useful in accordance with the presentinvention, is the combination of an aptamer recognizing a small compoundwith a hammerhead ribozyme. The conformational change induced in theaptamer upon binding the target molecule is supposed to regulate thecatalytic function of the ribozyme.

Hormones and cytokines are well known in the art. They are polypeptidesthat function as chemical messengers released by a cell or a gland inone part of the body sending out messages that affect cells in otherparts of the organism, thus transporting a signal from one cell toanother. Non-limiting examples for hormones and cytokines that might besuitable as test compounds include betatrophin, myokines (i.e. frommuscles), hepatokines, adipokines, incretins or other known cytokines(Lickert 2013).

Preferably, the screening methods of the present invention are carriedout in high-throughput format. High-throughput assays, independently ofbeing biochemical, cellular or other assays, generally may be performedin wells of microtiter plates, wherein each plate may contain 96, 384 or1536 wells. Handling of the plates, including incubation at temperaturesother than ambient temperature, and bringing into contact of testcompounds with the assay mixture is preferably carried out by one ormore computer-controlled robotic systems including pipetting devices. Incase large libraries of test compounds are to be screened and/orscreening is to be effected within a short time, mixtures of, forexample 10, 20, 30, 40, 50 or 100 test compounds may be added to eachwell. In case a well exhibits biological activity, said mixture of testcompounds may be de-convoluted to identify the one or more testcompounds in said mixture giving rise to said activity.

Also encompassed herein are modified versions of these compounds. Inother words, the compounds identified by the screening methods of thepresent invention may result in the identification of a lead compound,which is subsequently optimized to arrive at a compound that may, forexample, be used in a pharmaceutical composition. Methods for theoptimization of the pharmacological properties of compounds identifiedin screens, the lead compounds, are known in the art and comprise amethod of modifying a compound identified as a lead compound to achieve:(i) modified site of action, spectrum of activity, organ specificity,and/or (ii) improved potency, and/or (iii) decreased toxicity (improvedtherapeutic index), and/or (iv) decreased side effects, and/or (v)modified onset of therapeutic action, duration of effect, and/or (vi)modified pharmacokinetic parameters (resorption, distribution,metabolism and excretion), and/or (vii) modified physico-chemicalparameters (solubility, hygroscopicity, color, taste, odor, stability,state), and/or (viii) improved general specificity, organ/tissuespecificity, and/or (ix) optimized application form and route by (i)esterification of carboxyl groups, or (ii) esterification of hydroxylgroups with carboxylic acids, or (iii) esterification of hydroxyl groupsto, e.g. phosphates, pyrophosphates or sulfates or hemi-succinates, or(iv) formation of pharmaceutically acceptable salts, or (v) formation ofpharmaceutically acceptable complexes, or (vi) synthesis ofpharmacologically active polymers, or (vii) introduction of hydrophilicmoieties, or (viii) introduction/exchange of substituents on aromates orside chains, change of substituent pattern, or (ix) modification byintroduction of isosteric or bioisosteric moieties, or (x) synthesis ofhomologous compounds, or (xi) introduction of branched side chains, or(xii) conversion of alkyl substituents to cyclic analogues, or (xiii)derivatisation of hydroxyl group to ketales, acetales, or(xiv)N-acetylation to amides, phenylcarbamates, or (xv) synthesis ofMannich bases, imines, or (xvi) transformation of ketones or aldehydesto Schiff's bases, oximes, acetales, ketales, enolesters, oxazolidines,thiazolidines or combinations thereof.

The various steps recited above are generally known in the art. Theyinclude or rely on quantitative structure-activity relationship (QSAR)analyses (Kubinyi (1992) “Hausch-Analysis and Related Approaches”),combinatorial biochemistry, classical chemistry and others (Holzgrabeand Bechtold 2000).

In a first step, a cell population comprising immature progenitor βcells is contacted with a test compound as defined above. The cellpopulation comprising immature progenitor β cells may be obtained fromembryonic or pluripotent stem or progenitor cells or may be a primarycell population obtained from pancreas, such as e.g. pancreatic explantcultures, or parts thereof such as e.g. the islets of Langerhans, or maybe an established immature β cell line, such as e.g. MIN6 (mouse) andINS-1E (rat) insulinoma cell lines, which are commonly used in thediabetes research field, as well as e.g. the immortalized human β celllines (Ravassard, Hazhouz et al. 2011). Moreover, the cell populationmay also be a tissue preparation, such as for example (an) isolated,intact islet(s) of Langerhans. Islets of Langerhans are mini-organsthat, e.g. in mouse, are present in a capsule. They can be isolated fromthe pancreas after mildly digesting the pancreas with collagenase toloosen the extra-cellular matrix and then performing a density gradientcentrifugation to isolate the islets, which are visible under adissecting microscope and can be hand-picked. The cells tissuepreparations can be from any animal of interest. Preferably, the cellsor tissue preparations are from a mammalian animal, such as e.g. arodent, preferably a mouse, a rat or a rabbit; a pig, a dog, a cat or aprimate. In particular islets isolated from pig are a promising tool, ashigher amounts of islets can be isolated from pigs as compared to mice.

Further, the screening assay of the present invention may also becarried out in vivo in a suitable non-human animal model. Preferably,said non-human animal model is a mammalian animal, such as e.g. arodent, preferably a mouse, a rat or a rabbit; a pig, a dog, a cat or anon-human primate. However, it is particularly preferred that thisscreening method of the present invention is an in vitro method.

The cell population may comprise any amount of immature progenitor βcells, as long as immature progenitor β cells are present in detectableamounts. Preferably, the cell population comprises at least 5% immatureprogenitor β cells, such as at least 10% immature progenitor β cells,more preferably at least 20% immature progenitor β cells, even morepreferably at least 30%, such as at least 40%, at least 50%, at least60%, at least 70% or even more preferably at least 80% immatureprogenitor β cells. It is even more preferred that the cell populationcomprises at least 90% immature progenitor β cells, more preferably atleast 95% immature progenitor β cells and even more preferably at least98% immature progenitor β cells. Most preferably, the cell populationcomprises 100% immature progenitor β cells, i.e. all of the β cells inthe cell population are immature progenitor β cells and no mature βcells are present in detectable amounts. In those cases where the cellpopulation does not comprise 100% immature β cells, it is preferred thatthe remaining cells are selected from mature β cells that have alreadydifferentiated, as well as other cells naturally present in tissuescomprising β cells, such as e.g. the pancreas. Such other cells includee.g. ductal cells, acinar cells, or other endocrine cells of the isletof Langerhans, such as e.g. α-, PP-, δ-, and ε-cells, and fibroblasts.Typically, however, when using primary islet cell preparations,non-endocrine contaminating cells disappear during culture and representless than 1% of the total cells of the cell population. More preferably,the cell population does not comprise any α-, PP-, δ-, and ε-cells. Mostpreferably, the cell population consists of immature progenitor β cells,i.e. all of the cells in the cell population are immature progenitor βcells and no other cells are present. Means and methods of enriching acell population to the desired degree of purity are well known in theart and include, without being limiting, manual picking of cells orautomated picking by use of a robot, laser-capture microdissection orFACS (fluorescence activated cell sorting). Such methods are describedfor example in (Murray 2007, Tung, Heydari et al. 2007).

The term “at least” as used herein, such as e.g. the term “at least 5%”or “at least one” refers to the specifically recited amount or numberbut also to more than the specifically recited amount or number. Forexample, the term “at least 5% immature progenitor β cells” encompassesalso at least 6%, at least 7%, at least 8%, at least 9% immatureprogenitor β cells and so on. Furthermore, this term also encompassesexactly 5%, exactly 6%, exactly 7%, exactly 8%, exactly 9% immatureprogenitor β cells and so on.

The presence of immature progenitor β cells in the cell population canbe determined without further ado, for example based on thecharacteristics of immature progenitor β cells provided herein above.For example, the presence of the markers Pdx1 and Nkx6.1 can be used todetermine the presence of immature progenitor β cells in the cellpopulation together with a determination of the absence of the novelbiomarker Fltp and/or the absence of the established marker Ucn3.Suitable staining methods for showing the presence or absence of suchmarkers, either in individual cells or in the entire cell population,are well known in the art. Furthermore, as shown in Example 6 below,immature progenitor β cells were found herein to show a significantenrichment of genes that associate with cell proliferation, actinbinding, Wnt/PCP-, TGF receptor-, G-protein coupled receptor- andERK-signaling transduction. These genes may therefore serve as anindicator for the presence of immature progenitor β cells, e.g. viaimmunohistological staining of the cells for the expression products ofthese genes, or via in situ hybridisation techniques etc.

Preferably, the cell population comprising immature progenitor β cellsused for the screening (also referred to herein as the “starting cellpopulation”) is a population comprising, preferably consisting of,Nkx6.1-positive/Fltp-negative cells.

After contacting this cell population with the test compound, thepresence or expression level of the biomarker Fltp is determined in theβ cells comprised in the cell population in a subsequent step of themethod of the invention.

With regard to an individual β cell, the presence of Fltp indicates thatthe β cell is a mature β cell, as detailed herein above. Accordingly,where the effect of the test compound on an individual β cell isanalysed, the absence of Fltp expression within such a cell prior tocontacting with the test compound and the subsequent presence of Fltpexpression within such a cell after contacting with the test compoundindicates that the test compound is suitable for differentiatingimmature progenitor β cells into mature β cells. Similarly, if all thecells originally present in the cell population employed in thescreening assay were immature β cells (i.e. 100% immature β cellswithout any mature β cells), this starting population would have lackedFltp expression completely. In those cases, the presence of Fltp aftercontacting with the test compound indicates that the cell population nowcomprises mature β cells, as these cells now express the novel biomarkerFltp. Therefore, the presence of Fltp in these cell populations aftercontacting with the test compound is indicative of a compound suitablefor differentiating immature progenitor β cells into mature β cells.Determining the presence or absence of Fltp can be accomplished inseveral ways, as described above.

In those cases where the starting cell population already containsmature β cells, said starting cell population already expresses Fltp,i.e. Fltp is already present in the cell population. Accordingly, it isrequired that the expression level of Fltp is determined aftercontacting with the test compound, instead of simply determining thepresence thereof. The expression level of Fltp after contacting with thetest compound is then compared with the expression level of Fltp priorto contacting with the test compound and an increase in said expressionlevel after contacting with the test compound is indicative of acompound suitable for differentiating immature progenitor β cells intomature β cells.

The term “expression level”, as used herein, refers to a value ofexpression of a particular molecule in a sample of interest. Theexpression level corresponds to the number of copies of the expressionproduct of the corresponding gene, either on a nucleic acid level (e.g.mRNA) or on the protein level. Methods for determining the expressionlevel of a molecule include the methods described herein above fordetermining the presence or absence of a molecule on the nucleic acidlevel or on the amino acid level, such as e.g. hybridization assays,nucleic acid amplification assays, immunohistochemical methods but alsoe.g. Western blotting or polyacrylamide gel electrophoresis inconjunction with protein staining techniques such as Coomassie Brilliantblue or silver-staining.

The term “increased expression level”, as used herein, refers to ahigher expression level of the biomarker of interest, i.e. Fltp, in thecells after treatment with the test compound as compared to theexpression level observed in a control sample. Such a control sample canbe e.g. the cell population prior to contacting with the test compoundor a cell population that is of the same origin, and that is identicalin its characteristics to the cell population used in the assay but nottreated with the test compound. It will be appreciated that for thecontrol sample to be identical in its characteristics to the cellpopulation used in the assay, said control sample is not furthercultured under conditions that could change these characteristics, e.g.by inducing differentiation of the cells.

For example, a starting cell population may be divided and one part isused for determining the expression level of Fltp prior to treatment andthe second part is used in this method of the invention. Determining theFltp expression level in the control sample may be carried out prior toperforming the present method of the invention, such that the determinedvalues may be used as a reference at later times whenever a cellpopulation is screened in accordance with the method of the presentinvention; or may be determined in parallel each time a cell populationis screened in accordance with the method of the present invention. Sucha reference value may also be determined only once and stored as astandard for all future tests.

Preferably, the term “increased expression level” relates to astatistically significant higher expression level of Fltp aftertreatment as compared to the expression level observed in the controlsample. A statistically significant difference is for example when theamount of Fltp expression differs by at least 2a, i.e. two-times thestandard deviation from the expression level obtained in the controlsample, wherein the control sample is derived in repeated determinationfrom multiple samples. More preferably, the expression level of Fltp isconsidered to be increased if it is at least 10% higher after contactingwith the test compound as compared to the expression level observed inthe control sample, such as for example at least 20% higher, at least30% higher, at least 40% higher, at least 50% higher, at least 75%higher, at least 100% higher (i.e. twice as high), at least 200% higher,at least 300% higher, at least 500% higher etc. Preferably, the effectof the test compound on the expression level of Fltp is determined aftertreatment with said test compound for at least about 5 minutes, such asfor at least about 10 minutes, more preferably at least about 30minutes, such as e.g. at least about 1 hour, even more preferably atleast about 2 hours, such as e.g. at least about 3 hours, at least about4 hours, at least about 5 hours, at least about 6 hours, even morepreferably at least about 12 hours and most preferably at least about 24hours.

The term “about”, as used herein, encompasses the explicitly recitedamounts as well as deviations therefrom of ±15%. More preferably, adeviation of ±10%, and most preferably of ±5% is encompassed by the term“about”.

It will be appreciated that similar considerations apply in those caseswhere the starting cell population contains cells other than β cells butthat are suspected to, or known to, express Fltp, such as e.g. α-, PP-,δ-, and ε-cells. Also in those cases, Fltp is already present in thestarting cell population. In these cases, it will have to be ensuredthat the presence or increased expression of Fltp after contacting withthe test compound is determined in β cells but not in the other celltypes or in the overall population. Means and methods to determine theexpression of Fltp specifically in β cells are well known in the art.For example, using immunohistological stainings for Fltp as well ascell-specific additional markers, or by sorting cells using e.g. FACS,it can be determined whether a change of Fltp expression (from absent topresent or from present to an increased amount of expression) indeedoccurs in the β cells of the cell population.

The present invention also relates to a method of identifying a compoundsuitable for preventing the de-differentiation of mature β cells, themethod comprising: (a) culturing a cell population comprising mature βcells in the presence of a test compound, wherein the cells are culturedunder conditions that induce the de-differentiation of said mature βcells; and (b) subsequently determining the expression level of thebiomarker Flattop (Fltp) in the β cells cultured in step (a), wherein anexpression level of Fltp determined in step (b) that is substantiallyidentical to the expression level of Fltp in the cell populationcomprising mature β cells prior to the culture in step (a) is indicativeof a compound suitable for preventing the de-differentiation of mature βcells.

This embodiment relates to a screening assay for the identification of(a) compound(s) capable of preventing the de-differentiating of mature βcells, thereby maintaining mature β cells in culture. The novelbiomarker Fltp is used as a biomarker to detect the maturation state ofthe β cells.

All definitions and preferred embodiments provided herein with regard tothe use of the biomarker Fltp, the method for distinguishing mature fromimmature β cells and the method of identifying a compound suitable fordifferentiating immature progenitor β cells into mature β cells applymutatis mutandis also to this screening method of the invention.

In accordance with this method of the invention, a compound is screenedfor that is capable of preventing the de-differentiation of mature βcells, i.e. a compound that maintains mature β cells in culture asmature cells.

In a first step, a cell population comprising mature β cells is culturedin the presence of a test compound. Cell culture conditions are chosenthat would normally, i.e. in the absence of the test compound, inducethe de-differentiation of said mature β cells.

General cell culture conditions as well as suitable cell culture mediaare well known in the art (e.g. Cooper 2000 “Tools of Cell Biology”;Turksen, 2004 “Animal cell culture”). Preferred conditions and media aredetailed below.

It is preferred in any of the cell culture conditions described hereinthat the medium is exchanged (i.e. refreshed) at appropriate intervalsthat can be determined by the skilled person without further ado, suchas e.g. every four days, more preferably every three days, such as e.g.every two days and most preferably the medium is exchanged for freshcell culture medium every 24 hours.

Suitable cell culture media include, without being limiting, RPMI-1640(e.g. Biochrom), Dulbecco's MEM (e.g. FG 1445, Biochrom), Basal lscoveMedium (e.g. F0465, Biochrom), MCDB 153 Basal Medium (e.g. F8105,Biochrom AG), William's Medium E (e.g. F 1115, Biochrom) containingadditives including, without being limiting, FCS Gold (e.g. A15-151, PAALaboratories GmbH), L-glutamine (e.g. M11-004, PAA Laboratories GmbH),antibiotic/antimycotic (e.g. A5955, Sigma-Aldrich GmbH), gentamycin(e.g. A2712, Biochrom), insulin (e.g. 19278, Sigma-Aldrich GmbH) and/oradditional additives known in the art. Particularly preferred culturemedia are, for example, RPMI 1640 with 1 to 10% FBS (heat inactivated 30min 56° C.) and 1× Pen/Strep (corresponding to 100 units/ml ofpenicillin and 100 μg/ml of streptomycin); or RPMI 1640 or DMEM:F12(1:1) with 5% FCS (heat inactivated 30 min 56° C.), 2 mM L-Glutamin, 1×Pen/Strep, 1 mM Sodium pyrovate, 0.1 mM β-Mercaptoethanol, 1×non-essential amino acids. Most preferably, the cells are cultured inislet culture medium. 500 ml of islet culture medium consists of RPMI1640, 1 to 10% FCS and 5 ml Pen/Strep (100×).

Preferably, the cells or islets are cultured at 37° C. It is alsopreferred that the cells are cultured at 37° C. until the cells haveformed a confluent monolayer covering the surface of the culture dish.Typically, this takes between 2 to 4 days. The cells are then detachedusing protease treatment, such as e.g. TrypLE (e.g. Gibco, 12563) andare then re-plated at a diluted density of between 1:2 to 1:5 with isletculture medium (consisting of RPM′ 1640, 1 to 10% FCS and 1× Pen/Strep)and cultured again until a confluent monolayer covering the surface ofthe culture dish has grown, and so on (see above).

The term “conditions that induce the de-differentiation of said mature βcells” refers to all cell culture conditions that result in the loss ofthe maturation state of the mature β cells in said cell population. Suchcell culture conditions include, without being limiting, any of theabove recited culture media with reduced amounts of serum as compared tostandard media, such as e.g. the islet culture medium described above;without exogenous cytokines and without hormones. Preferably, the mediumcontains less than 1% serum, such as e.g. less than 0.9% serum, morepreferably less than 0.8% serum and even more preferably less than 0.5%serum.

More preferably, such a medium would consist of only islet culturemedium consisting of RPMI 1640, less than 1% FCS as well as 1× Pen/Strepand additives selected from amino acids, antibiotics and antimycotics,Alternatively, the de-differentiation can be induced by culturing thecells in the presence of reduced amounts of oxygen, such as e.g. 5% ofoxygen. As a further alternative, compounds that activate the hedgehogsignaling pathway may be added to the cell culture medium, in order toinduce de-differentiation. Such compounds are well known in the art andinclude, without being limiting, ligands such as e.g. Shh, Ihh and Dhhas well as agonists, such as e.g. SAG (smoothened agonist). Furthermore,depletion of FoxO1 or Pdx1 or Foxa2 or any other β cell specifictranscription factor or of β cell maintaining cytokines may also beemployed in order to induce de-differentiation (Landsman, Parent et al.2011, Pan and Wright 2011, Talchai, Xuan et al. 2012, Puri, Akiyama etal. 2013, Gao, McKenna et al. 2014). Such a depletion can be achievedwithout further ado, e.g. by using RNA interference or depletingantibodies.

It will be appreciated that in the latter two alternatives, cellsculture conditions comprising serum, preferably between 1 to 10% ofserum, can be employed.

It is well known in the art how to test whether the cell cultureconditions induce the de-differentiation of mature β cells. For example,the loss of maturation, i.e. the de-differentiation, can be detected bythe loss of markers of mature cells, such as Fltp, either alone or incombination with other maturation markers, such as e.g. Ucn3, asdetailed above. In addition, the down-regulation of genes specificallyexpressed in mature β cells and the up-regulation of genes specificallyexpressed in immature progenitor β cells can be analysed, as detailedherein above, to confirm a de-differentiation of the cells.

The cell population comprising mature β cells may be a primary cellpopulation, such as isolated endocrine cells, obtained from pancreas, orparts thereof such as e.g. the islets of Langerhans. Moreover, the cellpopulation may also be a tissue preparation, such as for example (an)isolated, intact islet(s) of Langerhans. Further, this screening assayof the present invention may also be carried out in vivo in a suitablenon-human animal model. Preferably, said non-human animal model is amammalian animal, such as e.g. a rodent, preferably a mouse, a rat or arabbit; a pig, a dog, a cat or a non-human primate. For example,de-differentiation has been described in animal models (Talchai, Xuan etal. 2012). Accordingly, a test compound could be screened, or anidentified lead compound could be verified, in such a model. Mostpreferably, this screening method of the present invention is an invitro method.

The cell population may comprise any amount of mature β cells, as longas mature β cells are present in detectable amounts. Preferably, thecell population comprises at least 5% mature β cells, such as at least10% mature β cells, more preferably at least 20% mature β cells, evenmore preferably at least 30%, such as at least 40%, at least 50%, atleast 60%, at least 70% or even more preferably at least 80% mature βcells. For example, when explant cultures are directly used as the cellpopulation, the cell population typically comprises about 80% mature βcells It is also preferred that the cell population comprises at least90% mature β cells, such as at least 95% mature β cells, at least 98%mature β cells and most preferably, the cell population comprises 100%mature β cells, i.e. all of the β cells in the cell population aremature β cells and no immature progenitor β cells are present indetectable amounts. Such a degree of purity may e.g. be obtained bysorting the cell population in order to enrich for mature β cells, forexample by using FACS sorting.

In those cases where the cell population does not comprise 100% mature βcells, it is preferred that the remaining cells are selected fromimmature progenitor β cells that have not yet differentiated or havede-differentiated, as well as other cells naturally present in tissuescomprising β cells, such as e.g. the pancreas. Such other cells includee.g. ductal cells, acinar cells, or other endocrine cells of the isletof Langerhans, such as e.g. α-, PP-, δ-, and ε-cells, and fibroblasts.Typically, however, when using primary islet cell preparations, suchnon-endocrine cells disappear during culture and represent less than 1%of the total cells of the cell population. More preferably, the cellpopulation does not comprise any α-, PP-, δ-, and ε-cells. Mostpreferably, the cell population consists of mature β cells, i.e. all ofthe cells in the cell population are mature β cells and no other cellsare present. Means and methods of enriching a cell population to thedesired degree of purity have been described herein above.

In accordance with this method of the invention, this cell population iscultured under de-differentiating conditions and in the presence of atest compound.

Any of the compounds recited herein above can be assayed as a testcompound in accordance with this method of the present invention ofidentifying a compound suitable for preventing the de-differentiation ofmature β cells.

In a second step, the expression level of the biomarker Fltp isdetermined in the β cells cultured in step (a). The methods describedherein above can also be employed with regard to this embodiment. Inaddition, the considerations concerning the determination of Fltpexpression levels in mixed populations detailed herein above with regardto the method of identifying a compound suitable for differentiatingimmature progenitor β cells into mature β cells apply mutatis mutandisto this method of identifying a compound suitable for preventing thede-differentiation of mature β cells.

The expression level of Fltp determined in the second step of thismethod of the invention is then compared to the expression level of Fltpin the cell population comprising mature β cells prior to the culture instep (a), i.e. to control values of Fltp expression. As detailed abovefor the method of identifying a compound suitable for differentiatingimmature progenitor β cells into mature β cells, several possibilitiesexist in this regard. For example, the expression level of Fltp can bedetermined in the cell population, or a part thereof, directly prior tocarrying out the cultivation step (a). Alternatively, a starting cellpopulation may be divided and one part is used for determining theexpression level of Fltp prior to treatment and the second part is usedin step (a) of this method of the invention. Determining this controlexpression level of Fltp may be carried out prior to performing thepresent method of the invention, such that the determined values may beused as a reference at later times whenever a cell population isscreened in accordance with the method of the present invention; or maybe determined in parallel each time a cell population is screened inaccordance with the method of the present invention. Such a controlvalue may also be determined only once and stored as a standard for allfuture tests. As detailed herein above with regard to the method ofidentifying a compound suitable for differentiating immature progenitorβ cells into mature β cells, said control value is obtained from asample that needs to be identical in its characteristics to the cellpopulation used in the assay. Accordingly, it is understood that saidcontrol sample is not further cultured under conditions that changethese characteristics. The preferred time points of determining theexpression level of Fltp described above apply mutatis mutandis also tothis method.

If this step of comparison of the treated cell population with theuntreated cell population shows that the expression level of Fltp issubstantially identical, then this finding indicates that the compoundis suitable for preventing the de-differentiating of mature β cells.

In accordance with all embodiments of the present invention, theexpression level of Fltp is considered to be “substantially identical”,if the expression level between the two samples differs by less than10%, preferably by less than 5%, more preferably by less than 4%, suchas by less than 3%, such as by less than 2%, and more preferably by lessthan 1%. Even more preferably, the expression level between the twosamples differs by less than 0.5% and most preferably, by less than0.1%. Even more preferably, the expression level between the two samplesdiffers only by deviations caused by the limits of accuracy ofestablished detection methods, in which case the expression levels areconsidered to be identical.

As discussed herein above, recent findings have shown that β cellde-differentiation contributes to β cell failure in type 2 diabetes.Accordingly, this method of the present invention enables for theidentification of novel lead compounds that could prove useful in thetreatment type 2 diabetes.

In a preferred embodiment of this method of the invention, the methodcomprises providing a cell population comprising mature β cells and

-   (a) culturing one portion of said cell population comprising mature    β cells in the presence of a test compound, wherein the cells are    cultured under conditions that induce the de-differentiation of said    cells; and-   (b) culturing a second portion of said cell population comprising    mature β cells in the absence of a test compound, wherein the cells    are cultured under conditions that induce the de-differentiation of    said cells; and-   (b) subsequently determining the expression level of the biomarker    Flattop (Fltp) in    -   (i) the cells cultured in (a), and    -   (ii) the cells cultured in (b);

wherein an increased expression level of Fltp in the cells cultured instep (a) as compared to the expression level of Fltp in the cellscultured in step (b) is indicative of a compound suitable for preventingthe de-differentiating of mature β cells.

In another preferred embodiment of the method of identifying a compoundsuitable for differentiating immature progenitor β cells into mature βcells or the method of identifying a compound suitable for preventingthe de-differentiating of mature β cells, the test compound is acompound that activates planar cell polarity (PCP).

The conserved planar cell polarity (PCP) pathway regulates theorientation of cells and organelles within the plane of a tissue andthus critically determines the function of mature cells in the contextof an organ ((Seifert and Mlodzik 2007, Wang and Nathans 2007,Wallingford 2012). Activation of the non-canonical Wnt/PCP signalingpathway triggers the asymmetric localization of core PCP molecules andcauses cytoskeletal rearrangements to provide three-dimensional (3D)tissue polarity information for cells (Wallingford and Mitchell 2011).The core PCP pathway components Van Gogh (Vangl), Flamingo/Cadherin EGFLAG seven-pass G-type receptor (Fmi/Celsr), Frizzled (Fzd), Dishevelled(DVI), and Prickle (Pk) are conserved during evolution (Wallingford andMitchell 2011).

Activation of the Wnt/PCP signaling pathway can be achieved viastimulation of the Wnt receptors (Frizzled (Frz) receptors) andco-receptors (Niehrs 2012), such as Ror1 ROR2, RYK, MUSK, PTK7, Syndecanor Glypican, via either naturally occurring ligands, small moleculecompounds and/or antibodies having an agonistic effect on thesereceptors. Accordingly, compounds that activate planar cell polarity(PCP) can be divided into ligands, intracellular mediators and core PCPcomponents. For example, Wnt5a and Wnt11 as well as the atypicalcadherins Fat and Dachsous are naturally occurring ligands that act viaWnt receptors and co-receptors. In addition, anti-Ror1- andanti-Ror2-antibodies are known in the art that can act as activators.Antibodies against Wnt5a, Wnt11 and Ror1/2 are commercially available,for example from R&D and other suppliers. Dishevelled (DvI) and Prickle(Pk) are intracellular mediators, while Van Gogh (Vangl1/2) andFlamingo/Cadherin EGF LAG seven-pass G-type receptor (Fmi/Celsr1, 2 and3) are core PCP components. All these compounds are well known in theart.

Moreover, the Wnt/PCP pathway also activates intracellular moleculessuch as small Rho GTPases, DAAM, RHOA, RACI, JNK, ROCK1 and ROCK2(Seifert and Mlodzik 2007, Wallingford and Mitchell 2011, Niehrs 2012,Ezan and Montcouquiol 2013, Matis and Axelrod 2013). Accordingly,agonists of such intracellular PCP activators can also be employed astest compounds in the methods of the invention.

In addition, the term “a compound that activates planar cell polarity(PCP)” also refers to activators and inhibitors of components of thispathway, i.e. compounds that indirectly activate the Wnt/PCP signalingpathway by modulating the activity of one of its components. Forexample, it is known in the art that the PCP signaling pathway and theHippo signaling pathway, which integrates physical signaling to regulatecell growth, are interconnected via Fat (Lawrence and Casal 2013).Accordingly, compounds that activate the Hippo signaling pathway mightactivate downstream signaling molecules of the PCP pathway. For example,the polarity regulating transcription factor Foxa2, which is regulatedby Hippo signaling (Sawada, Nishizaki et al. 2005), is known to directlytarget Flattop (Weedon, Cebola et al. 2014), showing that Fltpactivation can also be induced via indirect transcription factors thatmediate e.g. polarity, PCP and Hippo signaling.

In a more preferred embodiment of the method of the invention ofidentifying a compound suitable for differentiating immature progenitorβ cells into mature β cells or the method of the invention ofidentifying a compound suitable for preventing the de-differentiating ofmature β cells, the compound that activates planar cell polarity (PCP)is an activator of the non-canonical Wnt/PCP pathway.

The term “activator”, as used herein, is defined as a compound enhancingthe activity of a target molecule, preferably by performing one or moreof the following effects: (i) the transcription of the gene encoding theprotein to be activated is enhanced, (ii) the translation of the mRNAencoding the protein to be activated is enhanced, (iii) the proteinperforms its biochemical function with enhanced efficiency in thepresence of the activator, and (iv) the protein performs its cellularfunction with enhanced efficiency in the presence of the activator.Accordingly, the term “activator” encompasses both molecules that have adirectly activating effect on the specific pathway but also moleculesthat are indirectly activating, e.g. by interacting for example withmolecules that negatively regulate (e.g. suppress) said pathway.Compounds suitable to achieve the effect described in (i) includecompounds modulating the transcriptional machinery and/or itsinteraction with the promoter of said gene and/or with expressioncontrol elements remote from the promoter such as enhancers. Compoundssuitable to achieve the effect described in (ii) comprise compoundsmodulating the translational machinery. Compounds suitable to achievethe effect described in (iii) modulate the molecular functions of theprotein to be activated. Compounds suitable to achieve the effectdescribed in (iv) include compounds which do not necessarily binddirectly to the target protein, but still modulate their activity, forexample by binding to and/or modulating the function or expression ofmembers of a pathway which comprises the target protein. These membersare preferably upstream of the protein to be activated within saidpathway.

Such compounds include, without being limiting, all of the above recitedcompounds, such as e.g. small molecules, antisense nucleic acidmolecules, siRNA, shRNA, miRNA, antibodies, aptamers, ribozymes orpeptides such as soluble peptides.

Preferably, the level of activity of the non-canonical Wnt/PCP pathwayin the presence of an activator is 10% more than the activity of thenon-canonical Wnt/PCP pathway in the absence of the activator, morepreferred, the level of activity is 25% more, such as 50% more. Yet morepreferred are activators enhancing the level of activity of thenon-canonical Wnt/PCP pathway to 75%, 80%, 90% or 100% more than theactivity of the non-canonical Wnt/PCP pathway in the absence of theactivator.

The term “non-canonical Wnt/PCP pathway” is well known in the art andhas been described ((Seifert and Mlodzik 2007, Wallingford and Mitchell2011, Niehrs 2012, Ezan and Montcouquiol 2013, Matis and Axelrod 2013).Non-canonical Wnt/PCP pathway ligands, including, for example Wnt5a andWnt11, bind to a Wnt/PCP receptor and a co-receptor including, forexample, Frz 1, 2, 3, 6, or Ror1/2, causing a signal to be transduced toproteins including, for example, small GTPases, Cdc42, RhoA, lnturned,Fuzzy, and in particular Flattop. This results in the activation ofPI3K, JNK, RHOA, ROCK1/2 and other kinases, which then leads tocytoskeletal arrangements.

Accordingly, the term “activator of the non-canonical Wnt/PCP pathway”refers to an activator of any one of the above recited molecules thatform part of this signalling pathway. Preferably, the activator of thenon-canonical Wnt/PCP pathway is selected from the group consisting ofan activator of Wnt5a, an activator of Wnt11, other Wnt ligandsactivating the PCP pathway, ligands that activate the co-receptorsROR1,2, RYK, MUSK, PTK7, Syndecan or Glypican, as well as antibodies oralternative binding molecules, such as those described herein above, orsmall molecules that act agonistic on Wnt receptors and co-receptors.

The present invention further relates to a method of differentiatingimmature progenitor β cells into mature β cells, the method comprising:inducing the expression of Fltp in a cell population comprising immatureprogenitor β cells.

The definitions and preferred embodiments provided herein above, inparticular with regard to immature progenitor β cells, mature β cells,cell populations comprising immature progenitor β cells anddifferentiation apply mutatis mutandis to this method of the presentinvention.

In accordance with this method, immature progenitor β cells aredifferentiated into mature β cells. This method may be performed in vivoor in vitro. Preferably, the method is an in vitro method.

In accordance with this embodiment, it is particularly preferred thatthe immature progenitor β cells are obtained from embryonic orpluripotent stem or progenitor cells.

β cell differentiation is achieved, in accordance with this method ofthe invention, by inducing the expression of Fltp in said cells. Meansof inducing the expression of Fltp are not particularly limited. Forexample, Fltp expression can be induced by culturing immature progenitorβ cells in the presence of an activator of the non-canonical Wnt/PCPpathway, as Fltp has been found in accordance with the present inventionto be a downstream effector gene of this pathway. Compounds that inducethe expression of Fltp are also referred to herein as “activators ofFltp expression”.

In addition, expression of Fltp can be induced by culturing immatureprogenitor β cells at a high density, such as e.g. 90% confluency orcell densities known to trigger contact inhibition and polarization. Bygrowing immature progenitor β cells at such high densities, thecell-cell contacts, induced polarization and contact inhibition betweenthese cells lead to maturation of the cells.

Mature β cells obtained in accordance with this embodiment of theinvention are particularly useful for therapeutic approaches, such ase.g. for β cell replacement therapy.

In a preferred embodiment of this method of differentiating immatureprogenitor β cells into mature β cells, the expression of Fltp in thecells is induced by culturing the cells in the presence of a compoundselected from the group consisting of Wnt5a, Wnt11, Wnt3a, at least oneactivator of the co-receptors ROR1,2, RYK, MUSK, PTK7, Syndecan andGlypican and/or a compound identified by the method of the invention ofidentifying a compound suitable for differentiating immature progenitorβ cells into mature β cells.

Wnt5a is well known in the art and has been described (Yang 2012,Baarsma, Konigshoff et al. 2013). Wnt5a for use in cell culture can beobtained commercially, e.g. from R&D System. Preferred amounts of Wnt5ato be employed are between about 50 and about 200 ng/ml, more preferablybetween about 70 and about 150 ng/ml, such as for example between about90 and about 120 ng/ml and most preferably the amount is about 100ng/ml.

Wnt11 is well known in the art and has been described (Baarsma,Konigshoff et al. 2013). Wnt11 for use in cell culture can be obtainedcommercially, e.g. from R&D System. Preferred amounts of Wnt11 to beemployed are between about 50 and about 200 ng/ml, more preferablybetween about 70 and about 150 ng/ml, such as for example between about90 and about 120 ng/ml and most preferably the amount is about 100ng/ml.

Wnt3a is well known in the art and has been described (Baarsma,Konigshoff et al. 2013). Wnt3a for use in cell culture can be obtainedcommercially, e.g. from R&D Preferred amounts of Wnt3a to be employedare between about 50 and about 200 ng/ml, more preferably between about70 and about 150 ng/ml, such as for example between about 90 and about120 ng/ml and most preferably the amount is about 100 ng/ml.

The term “activator of the co-receptors ROR1,2, RYK, MUSK, PTK7,Syndecan and Glypican” encompasses any activator of these co-receptors,i.e. it includes direct and indirect activators. Preferably, anactivator of one of these co-receptors is a ligand of the respectiveco-receptor to be activated. Ligands that activate the co-receptorsROR1,2, RYK, MUSK, PTK7, Syndecan and Glypican for use in cell cultureare well known and can be obtained commercially, e.g. from R&D.Preferred amounts of the ligands that activate the co-receptors ROR1,2,RYK, MUSK, PTK7, Syndecan and/or Glypican to be employed are between 50and about 200 ng/ml, more preferably between about 70 and about 150ng/ml, such as for example between about 90 and about 120 ng/ml and mostpreferably the amount is about 100 ng/ml.

The present invention further relates to a method of preventingde-differentiating of mature β cells, the method comprising inducing ormaintaining the expression of Fltp in mature β cells.

The definitions and preferred embodiments provided herein above, inparticular with regard to mature β cells, de-differentiation as well asthe induction of Fltp expression apply mutatis mutandis to this methodof the present invention.

In accordance with this method, the de-differentiation of mature β cellsis prevented, i.e. the cells are maintained in a mature, functionalstate. This method may be performed in vivo or in vitro. Preferably, themethod is an in vitro method. In the latter case, the method comprises:culturing mature β cells in vitro under conditions suitable to induce ormaintain the expression of Fltp in the cells.

Means of inducing the expression of Fltp have been defined herein above.It will be appreciated that these means lead to the maintenance of Fltpexpression in those cases where Fltp is already expressed.

In a preferred embodiment of the method of preventing de-differentiatingof mature β cells, the expression of Fltp in the cells is induced byculturing the cells in the presence of a compound selected from Wnt5a,Wnt11, Wnt3a, an activator of at least one of the co-receptors ROR1,2,RYK, MUSK, PTK7, Syndecan and/or Glypican and/or a compound identifiedby the method of the invention of identifying a compound suitable forpreventing the de-differentiating of mature β cells.

These compounds have been defined herein above.

The present invention further relates to a kit for distinguishing matureβ cells from immature progenitor β cells, the kit comprising: (a) meansfor determining the presence or absence of the biomarker Flattop (Fltp),and (b) instructions how to use the kit.

Whereas the term “kit” in its broadest sense does not require thepresence of any other compounds, vials, containers and the like otherthan the recited components, the term “comprising”, in the context ofthe kit of the invention, denotes that further components can be presentin the kit. Non-limiting examples of such further components includepreservatives, buffers for storage, enzymes etc.

Where several components in accordance with (a) are comprised in thekit, the various components of the kit may be packaged in one or morecontainers such as one or more vials. Consequently, the variouscomponents of the kit may be present in isolation or combination. Thecontainers or vials may, in addition to the components, comprisepreservatives or buffers for storage. In addition, the kit containsinstructions for use.

“Means for determining the presence or amount of the biomarker Fltp” arewell known in the art and include, without being limiting, antibodiesspecifically binding (i.e. without cross-reacting with unrelatedmarkers) to Fltp in accordance with the present invention; nucleic acidprobes for the detection of Fltp on the nucleic acid level, such as forexample nucleic acid probes specifically hybridising with parts orfull-length nucleic acid molecules (DNA as well as RNA) encoding Fltp;sequencing primers for the analysis and detection of specific sequencesof the DNA encoding Fltp, e.g. sequences containing mutations known tointerfere with the expression of Fltp; or amplification primers foramplifying transcribed nucleic acid molecules of Fltp.

Also encompassed by this embodiment is that the kit comprises furthermeans for determining the presence or amount of biomarkers or referencemarkers different from the biomarker of the present invention, i.e.Fltp.

Such biomarkers different from Fltp include, without being limiting,additional β cell maturation markers, such as for example Urocortin 3(Blum, Hrvatin et al. 2012), as well as general cell markers, such asNkx6.1, Insulin, Pdx1 or any other β cell specific marker.

The term “reference marker”, as used herein, refers to a marker that ispresent in β cells at substantially constant levels. In other words, theexpression level of a reference marker should not differ (apart fromdeviations caused by the limits of accuracy of established detectionmethods) between β cells of different maturation levels. Due to theessentially unchanged amounts of such reference markers in different βcell populations, they may be employed to normalise values of biomarkeramounts, e.g. by comparison between the expression levels of saidnon-changing reference marker with the expression level of the biomarkerof interest. Often, housekeeping genes are used as reference markers.Examples of reference markers include, without being limiting, GAPDH,RPLP0, PGK1, HSP90AB1, cyclophilin, actin, HPRT, RN18s and many more.Further examples are described in literature (Velculescu, Madden et al.1999, Eisenberg and Levanon 2003).

If the kit comprises such additional means for determining the presenceor amount of biomarkers or reference markers different from Fltp inaccordance with the present invention, it is preferred that at most10.000 such additional markers are comprised in the kit of theinvention. More preferably, at most 5.000, such as for example at most2.000 and more preferably at most 1.000 additional nucleic markers arecomprised in the kit of the invention. More preferably, at most 800,such as for example at most 600, more preferable at most 400, such asfor example at most 300, at most 200, at most 100 and more preferably atmost 80 additional markers are comprised in the kit of the invention.Even more preferably, at most 50, such as for example at most 40, morepreferable at most 30, such as for example at most 20, at most 10, atmost 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most3, at most 2 and yet more preferably at most 1 additional marker(s)is/are comprised in the kit of the invention. Also preferred is that thekit of the invention only comprises means for determining the presenceor amount of Fltp.

The present invention additionally relates to a pharmaceuticalcomposition for use in treating or preventing diabetes, wherein thepharmaceutical composition comprises (an) activator(s) of Fltpexpression. Furthermore, the present invention relates to a method oftreating or preventing diabetes, the method comprising administering apharmaceutical composition comprising (an) activator(s) of Fltpexpression to a subject in need thereof.

In accordance with the present invention, the term “pharmaceuticalcomposition” relates to a composition for administration to a patient,preferably a human patient. The pharmaceutical composition of theinvention comprises the compounds recited above. The pharmaceuticalcomposition of the present invention may, optionally and additionally,comprise a pharmaceutically acceptable carrier. By “pharmaceuticallyacceptable carrier” is meant a non-toxic solid, semisolid or liquidfiller, diluent, encapsulating material or formulation auxiliary of anytype. Examples of suitable pharmaceutical carriers are well known in theart and include sodium chloride solutions, phosphate buffered sodiumchloride solutions, water, emulsions, such as oil/water emulsions,various types of wetting agents, sterile solutions, organic solventsetc. Preferably the carrier is a parenteral carrier, more preferably asolution that is isotonic with the blood of the recipient. The carriersuitably contains minor amounts of additives such as substances thatenhance isotonicity and chemical stability. Such materials are non-toxicto recipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, succinate, acetic acid, and otherorganic acids or their salts; antioxidants such as ascorbic acid; lowmolecular weight (less than about ten residues) (poly)peptides, e.g.,polyarginine or tripeptides; proteins, such as serum albumin, gelatin,or further immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, mannose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium; and/or nonionicsurfactants such as polysorbates, poloxamers, or PEG.

Compositions comprising such carriers can be formulated by well-knownconventional methods. Generally, the formulations are prepared bycontacting the components of the pharmaceutical composition uniformlyand intimately with liquid carriers or finely divided solid carriers orboth. Then, if necessary, the product is shaped into the desiredformulation.

The components of the pharmaceutical composition to be used fortherapeutic administration must be sterile. Sterility can for example beaccomplished by filtration through sterile filtration membranes (e.g.,0.2 micron membranes).

These pharmaceutical compositions can be administered to the subject ata suitable dose. The dosage regimen will be determined by the attendingphysician and clinical factors. As is well known in the medical arts,dosages for any one patient depend upon many factors, including thepatient's size, body surface area, age, the particular compound to beadministered, sex, time and route of administration, general health, andother drugs being administered concurrently. The therapeuticallyeffective amount for a given situation will readily be determined byroutine experimentation and is within the skills and judgment of theordinary clinician or physician. The pharmaceutical composition may befor administration once or for a regular administration over a prolongedperiod of time. Generally, the administration of the pharmaceuticalcomposition should be in the range of for example 10 μg/kg of bodyweight to 2 g/kg of body weight for a single dose. However, a morepreferred dosage might be in the range of 100 μg/kg to 1.5 g/kg of bodyweight, even more preferably 1 mg/kg to 1 g/kg of body weight and evenmore preferably 5 mg/kg to 500 mg/kg of body weight for a single dose.The length of treatment needed to observe changes and the intervalfollowing treatment for responses to occur vary depending on the desiredeffect. The particular amounts may be determined by conventional testswhich are well known to the person skilled in the art.

Administration of pharmaceutical compositions of the invention may beeffected by different ways, e.g., by parenteral (e.g. includingintravenous, intramuscular, intraperitoneal, intrasternal, subcutaneousand intraarticular injection as well as infusion), intradermal,intranasal or intrabronchial administration.

The components of the pharmaceutical composition ordinarily will bestored in unit or multi-dose containers, for example, sealed ampoules orvials, as an aqueous solution or as a lyophilized formulation forreconstitution. As an example of a lyophilized formulation, 10-ml vialsare filled with 5 ml of sterile-filtered 1% (w/v) aqueous solution, andthe resulting mixture is lyophilized. The infusion solution is preparedby reconstituting the lyophilized compound(s) using bacteriostaticwater-for-injection. Preservatives and other additives may also bepresent such as, for example, antimicrobials, anti-oxidants, chelatingagents, and inert gases and the like. The pharmaceutical composition maycomprise further agents depending on the intended use of thepharmaceutical composition. Since the pharmaceutical preparation of thepresent invention relies on the above mentioned compounds, it ispreferred that these mentioned further agents are only used as asupplement, i.e. at a reduced dose as compared to the recommended dosewhen used as the only drug, so as to e.g. reduce side effects conferredby the further agents.

A definition of the term “activator of Fltp expression”, usedinterchangeably with the term “inducer of Fltp expression”, has beenprovided herein above and applies mutatis mutandis also to thepharmaceutical composition of the present invention.

It is well known in the art that one of the results ofgluco-lipotoxicity is that β cells die and the architecture of the isletof Langerhans changes due to inflammation. In addition to stoppinginflammation, the restoration of the tissue architecture is therefore apromising approach to regenerate β cell functionality in the islet ofLangerhans. Activators of Fltp expression, as shown herein, provide avaluable tool to restore tissue polarity and enhance the maturation of βcells, thereby restoring the functionality of the islet of Langerhans.

In a preferred embodiment of the pharmaceutical composition of theinvention, the activator of Fltp expression is selected from the groupconsisting of Wnt5a, Wnt11, Wnt3a, an activator of at least one of theco-receptors ROR1,2, RYK, MUSK, PTK7, Syndecan and Glypican and/or acompound identified by the method of the invention of identifying acompound suitable for differentiating immature progenitor β cells intomature β cells. These compounds have been defined herein above.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. In case of conflict, thepresent specification, including definitions, will control.

As regards the embodiments characterized in this specification, inparticular in the claims, it is intended that each embodiment mentionedin a dependent claim is combined with each embodiment of each claim(independent or dependent) said dependent claim depends from. Forexample, in case of an independent claim 1 reciting 3 alternatives A, Band C, a dependent claim 2 reciting 3 alternatives D, E and F and aclaim 3 depending from claims 1 and 2 and reciting 3 alternatives G, Hand I, it is to be understood that the specification unambiguouslydiscloses embodiments corresponding to combinations A, D, G; A, D, H; A,D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B,D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C,D, G; C, D, H; C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C,F, I, unless specifically mentioned otherwise.

Similarly, and also in those cases where independent and/or dependentclaims do not recite alternatives, it is understood that if dependentclaims refer back to a plurality of preceding claims, any combination ofsubject-matter covered thereby is considered to be explicitly disclosed.For example, in case of an independent claim 1, a dependent claim 2referring back to claim 1, and a dependent claim 3 referring back toboth claims 2 and 1, it follows that the combination of thesubject-matter of claims 3 and 1 is clearly and unambiguously disclosedas is the combination of the subject-matter of claims 3, 2 and 1. Incase a further dependent claim 4 is present which refers to any one ofclaims 1 to 3, it follows that the combination of the subject-matter ofclaims 4 and 1, of claims 4, 2 and 1, of claims 4, 3 and 1, as well asof claims 4, 3, 2 and 1 is clearly and unambiguously disclosed.

The above considerations apply mutatis mutandis to all appended claims.To give a non-limiting example, the combination of claims 5 and 7 isclearly and unambiguously envisaged in view of the claim structure. Thesame applies for example to the combination of claims 5, 7 and 8, etc.

The figures show:

FIG. 1: Fltp reporter expression correlates with post-natal β cellmaturation and shows heterogeneity in all endocrine lineages.

(A) Representative images of single immunofluorescence stainings ofpancreatic tissue for Fltp reporter and Nkx6.1 on postnatal day 1 (P1)as well as of 12 weeks old Fltp^(ZV/+) animals. (B) Quantitativeanalysis of Fltp reporter expression in β cells during β cell maturationperiod in Fltp^(ZV/+) mice. Values are mean±SEM (standard error of themean); n=2 for P1, 3, 11, and n=9 for 12 weeks; ***P<0.001 in Fltpreporter and Nkx6.1 positive β-cells of P1 vs. 12 weeks old animals. Thepercentage of Fltp reporter expressing β cells increase from 45% (P1)over 70% (P11) to 80% (12 weeks). (C) Representative images of singleimmunofluorescence stainings of 4′,6-diamidino-2-phenylindole (DAPI),Fltp reporter, and one endocrine hormone on adult pancreatic tissue.Hormones are arranged from upper to lower row as indicated (glucagon,insulin, somatostatin, and pancreatic polypeptide). (D) Quantitativeanalysis of Fltp reporter expression reveals heterogeneity in Fltpreporter expression in all endocrine cell types (α=47.4%, β=81.1%,δ=49.3%, PP=47.0%). Values are mean±SEM; n>30 islets. Nuclear reporterof Fltp expression (Fltp) is marked by GFP antibody (A,C).

FIG. 2: β cell subpopulations exhibit differences in proliferativecapacity in vivo.

(A) Quantitative analysis of Ki67 expression in β cells upon metabolicdemand (pregnancy) in Fltp^(ZV/+) mice. Values are mean±SEM; n=4 forcontrol and pregnant mice embryonic day 15.5 (E15.5); **P<0.01 in Ki67expression in β cells which lack Fltp reporter expression vs. Fltpreporter expressing β cells in pregnant (E15.5) mice. (B) Representativeimages of single immunofluorescence stainings of Fltp reporter, Nkx6.1and Ki67 on adult pancreatic tissue of the control group and pregnantmice E15.5. (C) Quantitative analysis of β cell proliferation uponmetabolic demand (pregnancy) after 24 h in vivo pulse labeling using EdUin Fltp^(ZV/+) mice. Values are mean±SEM; n=5 for control and pregnantmice; ***P<0.001 in EdU marked β cells of pregnant mice (E15.5) whichlack Fltp reporter expression vs. Fltp reporter expressing β cells ofpregnant mice and vs. Fltp reporter negative β cells in control group.**P<0.01 in EdU marked β cells of pregnant mice (E15.5) expressing Fltpreporter vs. Fltp reporter expressing β cells of control mice. *P<0.05in EdU marked β cells of control mice which lack Fltp reporterexpression vs. Fltp reporter expressing β cells of control mice. (D)Representative images of single immunofluorescence stainings of Fltpreporter, Nkx6.1, and EdU on adult pancreatic tissue of the controlgroup and pregnant group at E15.5. (E) Quantitative analysis of Ki67expression in β cell upon metabolic demand (pregnancy) in Fltp^(ZV/+)mice. Values are mean±SEM; n=2 for P1, 3, 11 and n=4 for control andpregnant mice (E18.5); *P<0.05 in Ki67 expression in β cells which lackFltp reporter expression vs. Fltp reporter expressing β cells in P1, P3,P11 and pregnant (E18.5) mice. (F) Qualitative analysis of Fltp reporterexpression in β cells upon metabolic demand (pregnancy E15.5). Valuesare mean±SEM, n=5 for control and pregnant group; **P<0.01 betweenpercentage of Fltp reporter expressing β-cells compared to Fltp reporternegative β cells. Nuclear reporter of Fltp expression (Fltp) is markedby GFP antibody (B,D).

FIG. 3: Fltp^(ZV/ZV) β cells show 1^(st) phase insulin secretion defectsin vivo.

(A) Fltp^(ZV/+) as well as Fltp^(ZV/ZV) do not reveal altered glucosetolerance compared to Fltp^(+/+). The ability of Fltp^(ZV/+),Fltp^(ZV/ZV) and Fltp^(+/+) to handle a glucose load was assessed byusing a standard GTT. Fasted male mice were injected i.p. with glucose(2 g/kg of body weight), and blood glucose levels were measured at 0,15, 30, 60, and 120 min after glucose injection. (B) Quantitativeanalysis of total pancreatic insulin content in Fltp^(+/+), Fltp^(ZV/+)and Fltp^(ZV/ZV) mice shows no major difference (n=5 per genotype).Values are mean±SEM. (C) Quantitative analysis of first phase insulinsecretion reveals differences between Fltp^(ZV/ZV) and Fltp^(+/+) (n=4mice per genotype). Values are mean±SEM. (D) Quantitative analysis ofsecond phase insulin secretion shows no major difference. Values aremean±SEM.

FIG. 4: FLIP intronic SNP rs7515334 significantly associates withinsulin secretion defects in human.

(A) Depending on the metabolic status, the minor C1ORF192 allelers7515334 associates with increase insulin secretion (BMI <25) anddecreased insulin secretion (BMI >35) (B). Insulin secretion indexadjusted for gender, age, and OGTT-derived insulin sensitivity. Genotypetested in the dominant inheritance model with XG=CG+GG.Bonferroni-corrected α-level: p<0.0073.

FIG. 5: Fltp^(ZV/ZV) mice reveal altered susceptibility to STZ induced βcell death.

(A) Representative images of a single immunofluorescence stainings ofDAPI, Fltp reporter and Nkx6.1 on adult pancreatic tissue fromFltp^(ZV/+) mice treated with streptozotocin (STZ), Fltp^(ZV/+) vehicle(citrate buffer) (control), Fltp^(ZV/ZV) mice treated with STZ, andFltp^(ZV/ZV) vehicle (control). (B) After an overnight fast (16 h),Fltp^(ZV/+) and Fltp^(ZV/ZV) both treated with STZ and their respectivecontrols, mice were injected i.p. with glucose (2 g/kg of body weight)and blood glucose levels were measured at 0, 15, 30, 60, and 120 minafter injection. Fltp^(ZV/ZV) mice reveal an enhanced susceptibility toSTZ induced β cell damage and develop insulin resistance at an earliertime point compared to Fltp^(ZV/+). Values are mean±SEM; n=4 for STZmice and n=4 for control mice. *P<0.05 vs Fltp^(ZV/+). (C) Whole-bloodglucose concentration in Fltp^(ZV/+) and Fltp^(ZV/ZV) both treated withSTZ-induced diabetic mice (40 mg/kg, 5 days) and their respectivecontrol mice. Nuclear reporter of Fltp expression (Fltp) is marked byGFP antibody (A).

FIG. 6: Fltp reporter negative and positive islet subpopulations showdistinct gene expression differences.

(A) Schematic overview of experimental approach starting from isletisolation to Fluorescent activated cell sorting (FACS) to geneexpression analysis using microarray. (B) FACS plot of endocrinesubpopulations with side scatter (SSC) on y-axis and emission of 488 nmfluorescence on x-axis from isolated mouse islets of Fltp^(ZV/+) mouse(12 weeks old). (C) Representative images of single immunofluorescencestainings of Nkx6.1 and Fltp reporter on sorted Fltp reporter negativecells and Fltp reporter expressing cells. (D) Microarray analysis ofFltp mRNA expression in FACS sorted samples; two dimensional heat map ofmicroarray transcriptional gene profile containing genes which aredifferential regulated and important in endocrine cell function,polarity, signaling and cell cycle; P<0.05 and 1.5× regulated, 3 samplesleft are Fltp reporter expressing samples and 2 samples right are Fltpreporter negative samples. (E) GO-term enrichments after analysis withGeps of Genomatix®; bars represent significance by P-value. Nuclearreporter of Fltp expression (Fltp) is marked by GFP antibody (C).

FIG. 7: Genetic lineage tracing reveals that Fltp negative progenitorsgive rise to Fltp expressing mature β cells

(A) Scheme of the Fltp^(T2A-iCre/+) mTmG^(+/−) model. In this model allcells express membrane bond tomato (mT) except for the Fltp^(T2A-Cre)expressing cells which express membrane bound GFP (mG). When a Fltpnegative cell starts to activate the Fltp promoter the cells switchthrough a yellow state (mG and mT expression) into the green state (mGexpression). (B) Fluorescent activated cell sorting plot of islets fromFltp^(T2A-iCre/+)-mTmG^(+/−) model. Nkx6.1 staining control (up) andNkx6.1 stained cells (down). GFP and RFP expression are shown on theright (66.7% of Nkx6.1+ cell are GFP expressing cells, 12.3% are RFPpositive and 20% are GPF and RFP positive). (C) Representative imagesfrom live imaging experiment of islet isolated fromFltp^(T2A-iCre)-mTmG^(+/−) which shows the conversion of a mT expressingcell into mG and mT expressing state (white arrow).

FIG. 8: Fltp expression correlates with islet neogenesis and post-natalβ cell maturation.

(A-C′) Qualitative analysis of Fltp::H2B-Venus reporter activity inwhole-mount stained and BABB cleared pancreata and analyzed by LSManalysis at E18.5 and P5. Fltp reporter activity is detected by anti-GFPantibodies. High magnification images of compacted islets and cord-likestructures (C-C′). Fltp reporter activity strongly increases whileislets form and become compacted 3D structures, but is stillundetectable in cord-like structures.

FIG. 9: Fltp^(ZV/ZV) animals are Fltp null mutations.

(A) The Fltp^(ZV) targeting strategy deletes the whole open readingframe ranging from exon 2 (E2) till E6 (Primers for genotyping:5′-AGCCATACCACATTTGTAGAGG-3′, 5′-CAGCATGGCATAGATCTGGAC-3′,5′-GAGGCTGACTGGGAACAATC-3′). The external 5′- as well as the 3′-Southernprobe are indicated. Restriction enzyme sites for DraIII and EcoRV areshown. Homology regions for recombination of the targeting construct areindicated as 5′- and 3′-Retrieval (5′- and 3′-R). The figure is onscale. (B) Southern blot of WT embryonic stem (ES) cells versusFltp^(ZV/+) ES cells digested with DraIII and hybridized with theexternal 5′ Southern probe showing the BI6 (16443 bp) and 129 WT alleleas well as the BI6 targeted allele (11469 bp). Notice the shift of theWT band due to restriction length polymorphism. (C) Genotyping PCR todiscriminate between WT, Fltp^(ZV/+), and Fltp^(ZV/ZV) (Primers used:418, 565, and 566; WT band (317 bp); targeted Δneo band (387 bp)). (D)Western blot shows the absence of Fltp protein in testis lysate ofFltp^(ZV/ZV) animals. Fltp protein band is detectable at around 25 kDa(calculated weight 20 kDa). Abbreviations: NLS-LacZ: nuclearlocalization signal-beta-galactosidase; 2A: viral T2A sequence; H2B:histon-2B; Venus: yellow fluorescent reporter gene; SpA: Simian Virus 40polyadenylation signal; loxP: site of Cre mediate recombination; bpA:bovine Growth Hormone polyadenylation signal; neo: neomycin resistancecassette; PG: phospho-glycerate kinase; UTR: untranslated region; SP:Southern probe; VP: Venus probe.

FIG. 10: Generation a validation of the Fltp::Venus Fusion mouse line.

(A) The Fltp::Venus-Fusion targeting strategy fuses the open readingframe of Fltp to the fluorescent reporter gene Venus and the 3× FLAG tag(Primers for genotyping: 5′-CAGCATGGCATAGATCTGGAC-3′,5′-GAGGCTGACTGGGAACAATC-3′, 5′-CAAGATCCGCCACAACATCG-3′). The external5′- as well as the 3′ Southern-probe are indicated. Restriction enzymesites for DraIII are shown. Homology regions for recombination of thetargeting construct are indicated as 5′- and 3′-Retrieval (5′- and3′-R). (B) Southern blot analysis of targeted mouse ES cells (129Sv−/−C57Bl/6; IGD 3.2; Hitz et al., 2007). Genomic DNA was digested withDraIII and hybridized with the 5′ Fltp external probe resulting in bandsof 16443 bp (calculated size) for WT (Fltp^(+/+)) and 19133 bp forFltp::Venus Neo (Fltp^(V/+)) allele. (C) Genotyping PCR to discriminatebetween WT, Fltp^(+/+), and Fltp^(V/V) (Primers for genotyping:5′-CAGCATGGCATAGATCTGGAC-3′, 5′-GAGGCTGACTGGGAACAATC-3′,5′-CAAGATCCGCCACAACATCG-3′). WT band (317 bp); targeted Δneo band (430bp)). (D) Western blot analysis of lung and testis lysates of 2 monthsold male WT or Fltp^(+/+) animals using anti-Fltp antibody to detectendogenous Fltp (24 kDa) as well as the Fltp::Venus fusion protein (50kDa). Abbreviations: E1-6: exon 1-6; Venus: yellow fluorescent reportergene; bpA: bovine Growth Hormone polyadenylation signal; EM7: bacterialpromoter; loxP: site of Cre mediate recombination; neo: neomycinresistance cassette; PG: phospho-glycerate kinase; UTR: untranslatedregion; SP: Southern probe; VP: Venus probe; 3×F: 3× FLAG tag.

FIG. 11: Fltp with active Foxa2 binding sites in its promoter, antibodybinding sites and its conservation among species.

(A) Fltp shows Foxj1, Foxa1, and Foxa2 binding sites in its promoter(bar under the schematic gene). (B) Scheme of Fltp protein showing thetwo predicted proline rich repeats (PRRs) and the peptide sequences ofthe Fltp116-1 antibody as well as the Fltp1 antibody. (C, D) Westernblot showing the specificity of the Fltp116-1 (C) as well as the Fltp1(D) antibody in testis lysate of WT, Fltp^(ZVP), and Fltp^(ZV/ZV)animals. Fltp protein band is detectable at around 25 kDa (calculatedweight 20 kDa). (E) Immunohistochemistry on cryosections combined withLSM analysis to show that the Fltp116-1 antibody is specific. Fltp islocalized at the apical plasma membrane and along cilia inmulti-ciliated lung epithelial cells of WT (Ctrl) adult animals, but noFltp immunoreactivity is detected in Fltp^(ZV/ZV) lungs. Abbreviations:exonl-6 (E1-E6); TSS: transcriptional start site). Nuclei are marked by4′,6-Diamidin-2-phenylindol (DAPI), Fltp by Fltp116-1. Scale bars; 10μm.

FIG. 12: monoclonal antibodies against human FLTP

(A) Laser scanning microscopy (LSM) of EndoC-β H1 human β-cells stainedwith different monoclonal antibodies (clones #13, #28 and #43) againsthuman FLIP. As negative control human embryonic stem cells (hESCs) wereused (not shown). (B) Western Blot of Strep Flag-tagged human FLTPtransiently transfected in HEK293T and detected by monoclonal antibodies(clones #13, #28 and #43). Non-transfected HEK293T cells lysate wereused as negative control.

FIG. 13: FLTP mRNA expression in EndoC-β H1 human β-cells Significantincrease of human FLTP mRNA expression levels cultured under 3D vs 2Dconditions in EndoC-β Hβ cells is shown.

FIG. 14: 3D polarization and Wnt/PCP pathway activation in murine andhuman β-cells

(a,b) Laser scanning microscopy (LSM) of EndoC-β H1 human β-cellscultured in Matrigel (3D) show higher expression of β-cell andmaturation marker NKX6.1 compare to 2D conditions (b). Forquantification see d (3D: n=2; 2D: n=2). (c,g) Diagram showing thefluorescent intensity of Nkx6.1 in one single cell in 3D compared to 2D(c) and in 3D treated with Wnt5a and 3D without Wnt5a treatment (g).White lines indicate the measured region of interest (ROI) (i.e. in a,bfor c; e,f for g). (e,f) LSM of EndoC-β Hβ cells cultured Matrigel (3D)and treated with WNT5a show higher expression of NKX6.1 than under 3Dconditions (f). For quantification see h (WNT: n=1; control: n=4). (i,j)LSM of isolated islets of P5 WT animals either treated with (i) orwithout (j) Wnt5a for 3 days showing significant increase in Ucn3maturation marker staining. For quantification see I (Wnt5a treated:n=3; untreated: n=3). (k) Diagram showing the fluorescent intensity ofUcn3 of the re-aggregated islets+Wnt5a and without Wnt5a treatment.White lines in (i,j) indicate the measured ROI. (m) LSM picture ofEndoC-μ Hβ cells at day 7 showing compacted 3D pseudo-islets. (n) FLTPmRNA expression level in EndoC-βHβ cells cultured under 3D and 2Dconditions. Counting criteria for d,h are: The NKX6.1 fluorescentintensity of a certain number of cells of several independentexperiments were counted in 2D and 3D and the median fluorescentintensity was calculated with IMARIS. The median fluorescent intensitywas used as a threshold. All cells with higher intensity were counted asNKX6.1 high, all cells with lower intensity were counted as NKX6.1 lowcells. For FIG. 14d (3D: 300 NKX6.1⁺ cells; 2D: 221 Nkx6.1⁺ cells) werecounted. For FIG. 14h (WNT: 110 Nkx6.1⁺ cells; control: 550 Nkx6.1⁺cells) were counted. Scale bars, 20 μm (a,b,e,f), 10 μm (m).

FIG. 15: 3D polarization and Wnt/PCP pathway activation in murine andhuman β-cells (additional data)

(a,b) LSM of EndoC-β Hβ cells cultured in Matrigel (3D) show higherexpression of β-cell and maturation marker NKX6.1 than under 2D (b). Forquantification see d (3D: n=1; 2D: n=1). (c) Diagram showing thefluorescent intensity of Nkx6.1 in one single cell in 3D and in 2D.White lines in a,b show the measured region of interest (ROI). (e,f) LSMof isolated islets of P5 WT animals revealed a higher amount ofmaturation marker Ucn3 protein in islets treated with Wnt5a for 12 h (e)compared to non-treated islets (f). For quantification see h(islets+Wnt5a: n=3; untreated islets: n=3). (g) Diagram showing thefluorescent intensity of Ucn3 of the re-aggregated islets+Wnt5a andwithout Wnt5a. White lines in (e,f) indicate the measured ROI. Countingcriteria of c are: The NKX6.1 fluorescent intensity of a certain numberof cells of several independent experiments were counted in 2D and 3Dand the median fluorescent intensity was calculated with IMARIS. Themedian fluorescent intensity was used as a threshold. All cells withhigher intensity were counted as NKX6.1 high, all cells with lowerintensity were counted as NKX6.1 low cells. For FIG. 15d (3D: 571NKX6.1⁺ cells; 2D: 265 Nkx6.1⁺ cells) were counted. For FIG. 15v(islets+Wnt5a: 680 cells; untreated islets: 877 cells) were counted.Scale bars, 20 μm (a,b,e,f).

The examples illustrate the invention:

Example 1: Materials and Methods

Animal Studies

Mice were kept in the animal facility under optimal conditions in a 12-hlight cycle. Food and water were given ad libitum. Animal experimentswere carried out according to the German animal care and ethicslegislation and were approved by the local government authorities.

Eight-week-old Fltp^(ZV/+) females were paired to C57BL/6J males andseparated from the males after the appearance of the vaginal plugindicating day 0 of pregnancy. Pregnant females were euthanized on day15.5 of pregnancy (E 15.5) and the pancreas removed.

Glucose tolerance test (GTT) and insulin secretion test (IST) werecarried out in 12 weeks-old mice after a 12 h fast. Briefly, a singledose of glucose was intraperitoneally administrated (2 g/kg body weight)to the mice and blood glucose level was measured using Freestyle Liteglucometer (Abbot Laboratory, USA), at 0, 15, 30, 60 and 120 minutesfollowing glucose loading, by cutting off the tip of tail and squeezingit gently. For plasma insulin detection, blood sample were taken at 0,2.5, 5, 10 and 20 minutes following glucose loading (Andrikopoulos,Blair et al. 2008, Ayala, Samuel et al. 2010). Plasma insulin wasdetermined by using Ultra-sensitive mouse insulin ELISA kit (ChrystalChem, USA) according to the manufacturer's instructions.

For Streptozotocin (STZ)-mediated diabetes induction, freshly preparedSTZ (Sigma Aldrich, Germany) in 50 nM sodium citrate (pH 4.5) wasinjected intraperitoneally (40 mg/kg) daily for 5 days. Blood glucoselevel was measured every 2 days using Freestyle Lite glucometer (AbbotLaboratory). On day 16 after the first STZ injection, GTT was carriedout as described and mice were euthanized.

Generation of Animal Models

A Flattop-Venus fusion protein was generated by homologous recombinationin mouse embryonic stem (ES) cells by removing the translational stopcodon in exon 6 and directly fusing the Venus fluorescent protein to theopen-reading frame of Flattop. From these ES cells a knock-in mouse wasgenerated by germ line transmission of the targeted ES cells. These miceexpress the Flattop-Venus fusion protein in all tissues where Flattop isexpressed in equal amounts to the wild-type Flattop protein. ThisFlattop-Venus fusion reporter has the advantage that it is expressed inphysiological amount, shows normal protein turnover and shows normalsubcellular localization.

Antibody Generation

To be able to analyse the Fltp protein in more detail, two differentpolyclonal rabbit antibodies were raised against a central andC-terminal epitope (FIG. 11). The specificity of these antibodies wasconfirmed in western blot analysis and immunocytochemistry on lysatesand cells in which the Fltp gene was either over-expressed orknocked-out (FIG. 11). To be able to analyse the human FLIP protein inmore detail, three different monoclonal rat antibodies were raisedagainst a central and C-terminal epitope (FIG. 12). The specificity ofthese antibodies was confirmed in western blot analysis andimmunocytochemistry on lysates and cells in which the Strep-Flag taggedhuman FLTP cDNA was over-expressed (FIG. 12). These antibodies can beused as primary antibodies to detect the protein in tissues or cellcultures and using secondary antibodies either conjugated to horseradishperoxidase, alkaline phosphatase or fluorescent dyes.

Total Pancreatic Insulin Content

After 12 h starving, the animals were euthanized and the pancreasrapidly removed. Pancreas was placed into 5 ml 0.2 M HCl in 70% Ethanol,homogenized and incubated over night at −20° C. Subsequently, thehomogenized pancreas was again mixed with 0.2 M HCl in 70% Ethanol andincubated over night at −20° C. After centrifugation at 1000 g for 15minutes the supernatant was diluted (1:2) with 1 M Tris pH 7.5 and thenanalyzed. Insulin detection was performed by using Ultra sensitive mouseinsulin ELISA kit (Chrystal Chem, USA) according to the manufacturer'sinstructions. Total pancreatic protein content was estimated by Bradfordassay (Harlow and Lane 2006). Total pancreatic insulin content is statedas insulin (ng)/total pancreatic protein (μg).

LacZ Staining and Immunohistochemistry

Whole mount organ staining was performed as previously described (Huber,Kania et al. 2005). For whole mount imaging, embryos were cleared usingBABB (1 part benzyl alcohol, 2 parts benzyl benzoate). Forimmunohistochemistry staining, pancreas samples were fixed in 4%formalin, cryoprotected by incubation in sucrose gradient for 1 h each(5%, 15%, 30%) and embedded in Optimum Cutting Temperature (OCT). Cellssorted on glass slide were fixed with 2% PFA.

Nuclear staining was performed with DAPI (Life Technology, Germany). Forhistological assessment of islet β-cell proliferation, mice wereinjected with EdU Solution (100 μg/g of body weight) 24 h prior to beingsacrificed. EdU staining was performed using the Click-iT® Staining Kit(Life Technology) according to the manufacturer's instructions.Cryosection imaging was performed using Leica Confocal SP5 microscope.For quantification purposes, stained cells were counted manually onevery tenth section (14-15 μm thick frozen section). Quantification ofwhole mount organ staining was performed by using IMARIS software(Bitplane, Switzerland).

Islet Isolation, FACS Analysis and Gene Profiling

Islet isolation was carried out by collagenase P (Roche, Germany)digestion and centrifugation using Optiprep density gradient (Sigma).Isolated islets were handpicked two times under the microscope. After 1to 3 h of culture, islets were washed with PBS and incubate with 0.25%Trypsin-EDTA (Invitrogen) to obtain single cell suspensions. Singlecells were sorted using FACS-Aria III (BD Bioscience). The results wereanalyzed by using Flow Jo software. Total RNA was extracted usingmiRNeasy micro kit (Qiagen, Germany), amplified with the Ovation PicoSLWTA System V2 in combination with the Encore Biotin Module (Nugen, USA).Amplified cDNA was hybridized on Affymetrix Mouse Gene ST 1.0 arrayscontaining about 28,000 probe sets. Staining (Fluidics scriptFS450_0007) and scanning was done according to the Affymetrix expressionprotocol including minor modifications as suggested in the EncoreBiotion protocol. Expression console (v.1.3.0.187, Affymetrix) was usedfor quality control and to obtain annotated normalized RNA gene-leveldata (standard settings including median polish and sketch-quantilenormalization). Statistical analyses were performed by utilizing thestatistical programming environment R (R Development Core Team)implemented in CARMAweb. Genewise testing for differential expressionwas done employing the (paired) limma t-test and Benjamini-Hochbergmultiple testing correction (FDR <10%). Heatmaps were generated withCARMAweb and cluster dendrograms with the R script hclust. GO term andpathway enrichments were done for 1.5× regulated genes and aP-value<0.005 using the GePS module in the Genomatix Software Suite v3.1(Genomatix, Munich, Germany). For lineage tracing studies, islet fromeight-week-old Fltp^(T2A-Cre)-mTmG were isolated as described above. ForFACS analysis islet were incubate with 0.25% Trypsin-EDTA (Invitrogen)to obtain single cell suspensions and intracellular staining for Nkx6.1was performed. For live imaging experiment isolated islets fromFltp^(T2A-Cre)-mTmG were cultured in matrigel and imaging was performedusing Leica Confocal SP5 microscope.

Antibodies

Primary antibodies used for immunofluorescence: Goat anti-Nkx6.1 (R&Dsystem, Germany, AF5857 1:200); chicken anti-GFP (Ayes Labs, USA,GFP-1020 1:800); guinea pig anti-glucagon (Millipore, 4031-01F, 1:500);goat anti-somatostatin (Santa Cruz, USA, sc-7819, 1:300); rabbitanti-insulin (Thermo Scientific, USA, PA-18001, 1:300); guinea piganti-insulin (Thermo Scientific, USA, PA-26938, 1:300); goatanti-Pancreatic Polypeptide (PP) (Abcam, USA, ab77192, 1:300); rabbitanti-Ki-67 (Abcam, ab15580, 1:200); rabbit anti-Urocortin 3 (PhoenixPharmaceuticals, USA, H-019-29, 1:300); Alexa Fluor 546 phalloidin(Invitrogen, Germany, A22283, 1:200).

Secondary antibodies used for indirect fluorescence staining (dilution1:800 for all): Goat anti-chicken Alexa Fluor 488 (Dianova, Canada,103-545-155); donkey anti-goat Alexa Fluor 488 (Invitrogen, Germany,A11055); donkey anti-mouse Alexa Fluor 555 (Invitrogen, A31570); donkeyanti-goat Alexa Fluor 555 (Invitrogen, A21432); donkey anti-rabbit AlexaFluor 555 (Invitrogen, A31572); donkey anti-goat Alexa Fluor 594(Invitrogen, A11058); donkey anti-mouse Alexa Fluor 594 (Invitrogen,A21203); donkey anti-guinea-pig Alexa Fluor 649 (Dianova, 706-495-148).Nuclear staining was performed with DAPI (Life Technology, Germany). EdUstaining was performed using the Click-IT® Staining Kit (LifeTechnology) according to the manufacturer's instructions.

Statistical Analysis

Statistical analysis was performed using GraphPad Prism 6 Software(GraphPad Software, USA). Student's t-test was used for directcomparisons between two groups. A p value of <0.05 was considered asstatistically significant. Data is expressed as means±SEM/SD.

Example 2: The Expression of the Planar Cell Polarity (PCP) EffectorGene Fltp Strongly Increases During Post-Natal β Cell Maturation andShows Heterogeneous Levels in Adult Endocrine Cells

Three-dimensional (3D) and self-organized tissue architecture isrequired for organ formation and function (Eiraku, Takata et al. 2011,Sasai 2013). To determine whether the acquisition of tissue polarityduring islet neogenesis impacts on β cell function and maturation,tissue polarity establishment was analyzed on the molecular level byusing the Fltp::H2B-Venus reporter mice.

The Fltp reporter activity accurately reflects planar cell polarity(PCP) activity in the inner ear and lung, tissues that depend on Fltpfunction as a modulator of the actin and MT cytoskeleton dynamics. Fltpreporter activity was first analyzed at embryonic day (E) 18.5 when βcells are still organized in cord-like structures and vividly aggregateto form 3D sphere-like mini-organs. For this purpose, whole pancreatawere isolated, the tissue was cleared and laser-scanning confocalmicroscopy (LSM) analysis was used to acquire the entire 3D tissuedistribution of Nkx6.1⁺β cells (FIG. 8). Interestingly, Fltp reporterexpression was confined to 3 cells that were found in compacted islets,but was not detectable in β cells that were still organized in cord-likestructures. Additionally, it seemed that high levels of Nkx6.1 alsoinduced Fltp reporter activity, but only in already formed isletstructures. Together these results confirmed that Fltp reporter activityis switched on during planar cell polarity acquisition comparable to theinner ear and the lung.

To further investigate the expression of this PCP effector gene duringpost-natal β cell maturation (Blum, Hrvatin et al. 2012),Fltp::H2B-Venus reporter expression was analyzed in pancreatic sectionsshortly after birth (FIG. 1A). At postnatal day 1 (P1), Fltp reporteractivity was detected in less than 50% of Nkx6.1⁺ β cells. Reporteractivity rapidly increased in up to 70% of β cells while they matured innewly formed islets during the first ten days of life. In adult mice,Fltp reporter activity was detected in approximately 80% of β cells andaround 50% of other endocrine cell populations (FIG. 1C). These resultsshow that all endocrine cells express heterogeneous levels of PCPreporter activity. Additionally, the reporter activation during isletneogenesis and compaction likely reflects the changes in physicalproperties of the islet cell niche and might have a functional impact onall islet cells.

Example 3: Fltp Negative β Cells Show Increased Proliferative CapacityEspecially During β Cell Expansion Periods

For the further study, the focus was on the biological relevance of thePCP-related heterogeneity for β cells. Therefore, first theproliferation rate of Fltp reporter negative and Fltp reporterexpressing β cells during homeostasis, as well as duringpregnancy-induced and postnatal β cell expansion periods was compared.Strikingly, the Fltp reporter negative Nkx6.1⁺ β cells at P1, P3 and P11showed an up to four-fold higher replication rate when compared to Fltpreporter expressing Nkx6.1⁺ β cells, as measured by Ki67immunoreactivity in pancreatic cryosections (FIG. 2E). A similar up to4-fold difference in the proliferation rate of these two β cellsubpopulations was observed during pregnancy (FIGS. 2A and 2E). Theconcomitant and significant decrease of Fltp reporter positive cellsfrom 80% to 70% (FIG. 2F) indicates that mainly the Fltp reporternegative cells proliferate and contributed to β cell expansion duringpregnancy. Even in adult mice fed ad libitum, a physiological statewhere 6 cells are in homeostasis and refractory to proliferation, Fltpreporter negative β cells still showed an increased replication capacity(FIG. 2C). These proliferation studies were confirmed by EdU-pulselabeling during β cell homeostasis and expansion (FIG. 2C). Thuscollectively these data illustrate that the two subpopulations of βcells show markedly different proliferative capacity depending onenvironmental conditions and cell polarity status.

Example 4: Loss of Mouse Fltp and an Intronic SNP in Human FLTPAssociates with Insulin Secretion Defects

It is well established that β cells are functionally coupled and thatinsulin secretion depends on the actin and MT cytoskeleton (Kalwat andThurmond 2013). To analyze if the PCP effector protein Fltp is necessaryfor adult β cell function, a glucose tolerance test was performed usingintra-peritoneal glucose stimulation (ipGTT) in adult males on a chowdiet (FIG. 3A). Only a slight but not significant delay in glucoseclearance was observed in Fltp^(ZV/ZV) and Fltp^(ZV/+) mice, whencompared to Fftp^(+/+) littermates. Also, no significant difference intotal pancreatic insulin content was observed between Fltp^(+/+),Fltp^(ZV/+) and Fltp^(ZV/ZV) mice (FIG. 3B). Interestingly, 1^(st) (FIG.3C), but not 2^(nd) phase insulin secretion (FIG. 3D) seems to bedelayed in homozygous mutants when compared to Fltp^(+/+) littermates,suggesting that the PCP activity and Fltp function is necessary forglucose-induced insulin secretion of jβ cells.

To seek first evidence whether the human orthologue gene C1Orf192 isalso associated with metabolic traits and 10+ SNPs were screened forgenetic association in a cohort of 2100 human pre-diabetic and diabeticsubjects. Interestingly, this revealed that the intronic SNP rs75715534with a minor allele frequency of 0.2 significantly associated withincreased insulin secretion on lean subjects (BMI <25), whereas the sameSNP associated with decreased insulin secretion in obese subjects(BMI >35) (FIGS. 4A and B). These results suggest that human FLTPdifferentially reacts to metabolic demand and is generally important forglucose-induced insulin secretion. Together these data suggest thatFltp-dependent cytoskeletal rearrangements established during planarcell polarity are important for glucose-induced insulin secretion of βcells.

Association Analysis

Subjects.

The study population consisted of 2,228 Caucasians at risk for type 2diabetes (family history of type 2 diabetes, body mass index (BMI) 27kg/m², impaired fasting glycaemia, and/or previous gestational diabetes)recruited from the ongoing Tubingen Family study for type 2 diabetes(1). All participants underwent assessment of medical history, smokingstatus, and alcohol consumption habits; the subjects furthermore agreedto undergo physical examination, routine blood tests, and oral glucosetolerance tests (OGTTs). Only individuals with complete phenotypic andgenotypic data sets and documented absence of medication known toinfluence glucose tolerance, insulin sensitivity, or insulin secretionwere included. All study participants gave informed written consent tothe study which adhered to the Declaration of Helsinki.

The study protocol was approved by the OGTT and laboratory measurements.A standardized 75-g OGTT was performed following a 10-hovernight fast.For the determination of plasma glucose, insulin, and C-peptide levels,venous blood samples were drawn at baseline and at time-points 30, 60,90, and 120 min of the OGTT (Stefan, Machicao et al. 2005). Plasmaglucose levels (in mmol/L) were measured with a bedside glucose analyser(glucose oxidase method, Yellow Springs Instruments, Yellow Springs,Ohio, USA). Plasma insulin and C-peptide levels (in pmol/L, both) weredetermined by commercial chemiluminescence assays for ADVIA Centaur(Siemens Medical Solutions, Fernwald, Germany). BMI was calculated asweight divided by squared height (in kg/m²). OGTT-derived insulinsensitivity was estimated as proposed earlier (Matsuda and DeFronzo1999): 10,000/[c(Glc0)*c(Ins0)*c(Glcmean)*c(Insmean)]I (withc=concentration, Glc=glucose, and Ins=insulin). OGTT-derived insulinsecretion was estimated as area under the curve (AUC) Cpep0-30/AUCGlc0-30 according to the formula:[c(Cpep0)+c(Cpep30)]/[c(Glc0)+c(Glc30)] (with Cpep=C-peptide). EthicsCommittee of the Eberhard Karls University Tubingen.

Selection of Tagging SNPs and Genotyping.

Based on publicly available data from the 1000 Genomes Project(http://browser.1000genomes.org/index.html), we analysed in silico agenomic area on human chromosome 1q23.3 spanning the C1orf192 gene(3.143 kb, five exons, four introns, located on the reverse strand) and2 kb of the gene's 5′-flanking region. Within the analysed C1orf192locus, 36 SNPs were found. Using the tagger analysis tool of Haploview(see the Wordl Wide Web atbroadinstitute.org/scientific-community/science/programs/medical-and-population-genetics/haploview/haploview),seven tagging SNPs were identified that cover all the other common SNPs(minor allele frequency ≧0.01) with an r² 0.8. These SNPs werers17399583 (C/T), rs11584714 (C/G), and rs57835711 (C/G) in the5′-flanking region, rs114482063 (A/T) and rs182840301 (C/A) in intron 1,and rs16832872 (G/A) and rs75715534 (C/G) in intron 3. For genotyping,DNA was isolated from whole blood using a commercial kit (NucleoSpin,Macherey & Nagel, Duren, Germany). The tagging SNPs were genotyped usingthe mass spectrometry system massARRAY from Sequenom and themanufacturer's iPLEX software (Sequenom, Hamburg, Germany). The callrates were 96.1%. The mass spectrometric results were validated in 50randomly selected subjects by bidirectional sequencing, and both methodsgave 100% identical results.

Statistical Analyses.

Continuous variables with non-normal distribution werelog_(e)-transformed prior to statistical analysis. Multiple linearregression analysis was performed using the least-squares method. In theregression models, insulin secretion was chosen as outcome variable, theSNP genotype (in the dominant inheritance model) as independent variableand gender, age, BMI, and insulin sensitivity as confounding variables.SNP-BMI interaction effects on insulin secretion were tested by analysisof covariance (ANCOVA) with gender, age, and insulin sensitivity asconfounding variables. When testing all seven tagging SNPs in parallel,a Bonferroni-corrected p-value <0.0073 was considered statisticallysignificant. For all analyses, the statistical software JMP 8.0 (SASInstitute, Cary, N.C., USA) was used.

Example 5: Loss of Fltp Makes β Cells More Susceptible to StreptozotocinTreatment

To further test the requirement of PCP and Fltp in the context of β cellsurvival and regeneration, a multiple low-dose streptozotocin (STZ)model was employed (Kolb 1987). Streptozotocin is a naturally occurringchemical that is particularly toxic to β cells. For this purpose STZ wasinjected on five consecutive days and blood glucose regulation wasmeasured every 2^(nd) day thereafter in Fltp^(ZV/+) and Fltp^(ZV/ZV)cohorts (FIG. 5C). This revealed that blood glucose control and β cellfunction gradually decreased in a Fltp-dose dependent manner until day16 after the first injection. An ipGTT performed at day 16 after thefirst STZ injection revealed that Fltp^(ZV/ZV) mice were significantlymore glucose intolerant when compared to Fltp^(ZV/+) littermates (FIG.5B). The gradual loss of β cells was confirmed by immunohistochemistryon pancreatic sections from vehicle and STZ-treated animals (FIG. 5A),which at the stage analyzed did not show any signs of β cellregeneration. Thus, loss of Fltp and planar polarization in β cellsincreases the vulnerability to STZ.

Example 6: Molecular Profiling Reveals Increased Maturation State ofFltp Reporter Expressing β Cells

The data presented so far clearly indicated that Fltp-mediated planarpolarization subdivided β cells into proliferative and into more matureβ cells. To better understand these PCP-mediated differences on themolecular level, use was made of the Fltp::H2B-Venus reporter. Adultislets were purified from animals under physiological homeostaticconditions and the Fltp reporter negative and Fltp reporter expressingendocrine subpopulations were isolated using fluorescent activated cellsorting (FACS; FIGS. 6A&B). The purity of the Fltp reporter negative andFltp reporter expressing endocrine cell populations were controlled bycytospins and reached almost 100% for the Venus fluorescent marker andapprox. 80% of both populations were β cells (marked by Nkx6.1) (FIG.6C).

The Fltp reporter negative and Fltp reporter expressing endocrinesubpopulations showed markedly different levels of mRNA expression aftercDNA amplification and expression profiling using Affymetrix gene arrays(FIG. 6D). 1887 genes are up- and 1800 genes are more than 1.5-foldsignificantly (p<0.005) down-regulated. Strikingly, unbiased geneontology (GO) term analysis revealed that the Fltp reporter negativepopulation shows a significant enrichment of genes that associate withcell proliferation, actin binding, Wnt/PCP-, TGF receptor-, G-proteincoupled receptor-, ERK-signaling transduction, whereas the Fltp reporterexpressing population shows significant enrichment of genes that areimportant for mature beta cell function, such as genes involved inmetabolic processes, glucose metabolism, mitochondria and insulinsecretion (FIG. 6E).

Taken together, the molecular profiling of Fltp reporter negative andFltp reporter expressing endocrine populations clearly revealed thatFltp reporter negative cells constitute a population of low polarizedmore naïve progenitor cells, whereas the Fltp reporter negative cellsare highly polarized, more mature and express higher levels ofglycolysis enzymes, polarity markers and signaling receptors for major βcell regulatory pathways.

Example 7: Fltp Reporter Negative β Cell Progenitors Directly Give Riseto Fltp Reporter Expressing Mature Beta Cells

To directly test whether Fltp reporter negative β cells are progenitorsof Fltp reporter expressing β cells, a Cre recombinase/loxP-mediatedgenetic lineage tracing approach was used. To this end, the previouslyestablished Fltp-T2A-iCre mouse line (Lange, Gegg et al. 2012) wascrossed to the mTmG reporter mouse line (Muzumdar, Tasic et al. 2007).Upon Fltp promoter-driven Cre expression, the membrane Tomato (mT)fluorescent reporter gene switches to membrane GFP (mG), which isirreversible and therefore allows cell fate analysis. Islets of thesecrossings were isolated and cultured in vitro. To establish the cultureconditions, it is first tested if β cells depend on Wnt/PCP signaling tomaintain Fltp reporter expression. Therefore, islets were cultured inabsence or presence of ligands of the canonical Wnt/β-catenin (Wnt3a)and non-canonical Wnt/PCP pathway (Wnt5a). These experiments indicatedthat Fltp-reporter gene expression indeed depend on Wnt/PCP, but notcanonical Wnt signaling. Using these culture conditions in combinationwith live single cell imaging will allow us to follow new born Fltp-Creexpressing mT and mG expressing cells over a time-course of two to fourdays. Single-cell tracking over this time period and later fixation andstaining for the β cell marker Nkx6.1 clearly revealed that mTexpressing cells (Fltp negative) cells give rise to mG expressing cells(Fltp expression), suggesting that adult islets contain a Fltp negativelow polarized and highly proliferative progenitor population, which cangive rise to Wnt/PCP-dependent highly polarized and less proliferativeFltp expressing mature β cell population.

Example 8: Nkx6.1 Expressing β Cell Progenitors Form Cord-LikeStructures which Later Arrange in Islets of Langerhans

Analysis of Nkx6.1 expressing β cells in whole-mount stained and BABBcleared pancreata at E18.5 show cord-like structures. During post-natalmaturation period they become compacted 3D structures. The Fltp reporteris absent in b cells located in cord-like structures compared to islets.Together with the increased Fltp reporter expression during maturation(FIG. 1B) and the change in morphology of cord-like structures to isletsindicate that Fltp expression is connected with islets formation.

Example 9: Knock-in/Knock-Out of lacZ and H2B-Venus into the Fltp Locus

To explore Fltp expression and function in vivo, a Fltp^(ZV)knock-in/knock-out allele was generated where the entire ORF wasreplaced by a multicistronic lacZ-Venus reporter cassette. Thiscontained a nuclear localization signal (NLS)-tagged β-Galactosidase(lacZ) reporter gene followed by an intervening viral Thosea Asigma2A-peptide (T2A) for co-translational cleavage and a very bright Histone2B (H2B)-Venus fluorescent reporter gene. Southern and western blotanalysis confirmed the targeted homologous recombination and generationof a null allele.

The knock-in/knock-out construct was designed as shown in FIG. 9. 5′ and3′ HR for the Fltp gene were amplified by PCR (449 fwd 5′ HR Ascl:5′-NNNGGCGCGCCAGTCAGGAAGTGGAAGAGAAGAACACAG-3′; 450 rev 5′ HR HindIII,SpeI: 5′-NNNAAGCTTACTAGTGTGGTGGAGTGCCTGTCTACATGTG-3′; 451 fwd 3′ HRHindIII: 5′-NNNAAGCTICACGACAGTCAAAGCTGCAATAGAAC-3′; 452 rev 3′ HR BamHI:5′-NNNGGATCCGGTAATTTGGCAATTATAGAACTCAGGC-3′) using a C57BL/6J BAC clone(RP23-333P11) as template. These two PCR products were subcloned intothe pL254 vector (Liao, Uetzmann et al. 2009) using Ascl and BamHI. Theresulting vector was digested with HindIII, SpeI and electroporated intoelectrocompetent EL350 bacteria containing the Fltp BAC clone toretrieve the WT sequence between PCR homology arms resulting in the Fltpretrieval vector. For cloning of the knock-in/knock-out cassette inpBKS-5′ and 3′ HR for the knock-in into the ATG of exon two of Fltp weregenerated by PCR (453 fwd 5′ HR SacII:5′-NNNCCGCGGAGCAGACTTAACTATGTTGGGGAAACAGC-3′; 454 rev 5′ HR SalI, NotI:5′-NNNGTCGACGCGGCCGCTGTTTACACTTGTTGCCTGGCAACTG-3′; 455 fwd 3′ HR SalI:5′-NNNGTCGACGGTCCTAGTCTAGCTGAGGTCCAGATC-3′; 456 rev 3′ HR KpnI:5′-NNNGGTACCATGCTGTGGGAGTCACTGACATTCTTG-3′) using the previouslymentioned BAC as a template and subcloned into pBKS-using the introducedrestriction sites, resulting in pBKS-Fltp-HomArms. The first step togenerate the targeting vector was to construct thepBKS-H2B-Venus-intron-SV40pA plasmid by subcloning an oligonucleotidefor the H2B (histone 2B) that introduces a 5′ NotI and a perfect Kozaksequence (025: 5′-NNNGCGGCCGCGCCACCATGCCAGAGCCAGCG-3′) and a 3′ XbaIsite (026: 5′-NNNTCTAGACTTAGCGCTGGTGTACTTGGTGATGG-3′). This PCR productwas ligated into pBKS-(both cut with NotI, XbaI) resulting in pBKS-H2B.The next step was to introduce the Venus reporter gene (yellowfluorescent protein) also via PCR with the forward (fwd) primercontaining an XbaI site (013:5′-NNNTCTAGAATGGTGAGCAAGGGCGAGGAGCTGTTC-3′) and a reverse (rev) primercontaining a SpeI site (014:5′-NNNACTAGTTTACTTGTACAGCTCGTCCATGCCGAGAG-3′). This PCR and the vectorpBKS-H2B were digested with XbaI and SpeI and ligated resulting inpBKS-H2B-Venus. To complete the construct an intron-SV40pAoligonucleotide was generated by using the fwd primer containing SpeI(011: 5′-NNNACTAGTAGGTAAGTGTACCCAATTCGCCCTATAG-3′) and the rev primercontaining BamHI (012: 5′-NNNGGATCCACGCGTTAAGATACATTGATGAGTTTGGAC-3′).This oligonucleotide was subcloned into pBKS-H2B-Venus by cutting bothwith SpeI and BamHI resulting in the pBKS-H2B-Venus-intron-SV40pAplasmid. The next step was to introduce the loxP flanked neomycin (neo)resistance cassette by digesting the PL-452 vector (Liu, Jenkins et al.2003) with SalI and BamHI. The digested vector was ligated into thepBKS-H2B-Venus-intron-SV40pA plasmid opened by cutting with SalI andBamHI resulting inpBKS-H2B-Venus-intron-SV40pA-loxP-bGHpA-neo-EM7-PGK-loxP(pBKS-H2B-Venus-neo). For following cloning steps it was necessary todestroy the MluI site located in the SV40pA by cutting with MluI,filling up the 5′ overhang with Klenow polymerase and religating thevector. The T2A sequence from Thosea asigna virus was introduced intothe NotI site of pBKS-H2B-Venus-neo by annealing the following oligos2A_fwd (5′-GGCCGCACGCGTTTGAAGGTAGAGGCTCTTTACTAACATGCGGCGACGTTGAGGAAAACCCAGGACC-3′) and 2A_rev(5′-GGCCTGGTCCTGGGTTTTCCTCAACGTCGCCGCATGTTAGTAAAGAGCCTCTACCTTCAAACGCGTGC-3′), which created a NotI compatible overhang resulting inpBKS-2A-H2B-Venus-neo. To clone the NLS-lacZ (nuclear localisationsignal-b-galactosidase fusion protein) in front of the H2B-Venusconstruct we amplified the NLS-lacZ by PCR out of a NLS-lacZ containingvector. We used the fwd primer 340(5′-NNNGCGGCCGCGCCACCATGAACCTTGAAGCTCGAAAAACAAAG-3′) with a NotI site atthe 5′ end and the rev primer 341(5′-NNNGGCGCGCCTTTTTGACACCAGACCAACTGGTAATGGTAGC-3′), containing an Asclsite at the 3′ end. The PCR product was digested with NotI and Ascl andligated into the NotI and MluI digested pBKS-2A-H2B-Venus-neo vectorresulting in pBKS-NLS-lacZ-2A-H2B-Venus-neo. For finishing theminitargeting construct we cloned pBKS-NLS-lacZ-2A-H2B-Venus-neo intopBKS-Fltp-HomArms (both cut with NotI and SalI). The minitargetingconstruct was cut out by SacII and KpnI, electroporated in EL350bacteria and introduced into PL254 via bacterial homologuesrecombination resulting in the final targeting construct(PL254-Fltp-NLS-lacZ-2A-H2B-Venus-intron-SV40pA-loxP-bGHpA-neo-EM7-PGK-loxP)which was confirmed by sequencing and is ready for electroporating intoembryonic stem (ES) cells (after linearization by Ascl).

Example 10: Fltp-Venus Fusion Knock-in Strategy and Confirmation

The knock-in construct was designed as shown in FIG. 10. The Fltpretrieval vector was generated as described in example 9.

For cloning of the knock-in cassette in pBKS—5′ and 3′ HR for theknock-in into the ATG of exon two of Fltp were generated by PCR withfollowing primers: 1228 fwd 5′ HR:5′-NNNGCGGCCGCGGTTGGATTCTGAGGCTGACTGGG-3′, and 1229 rev 5′ HR:5′-NNNTCTAGACTTGGTGCTCTTACAAGGGCTCGG-3′, digested with NotI and XbaI;EP1230 fwd 3′ HR: 5′-NNNGAATTCGTCCTAGTCTAGCTGAGGTCCAGATCTATG-3′, andEP1231 rev 3′ HR: 5′-NNNAAGCTTGTGGGAGTCACTGACATTCTTGTTAACC-3′, digestedwith EcoRI and HindIII using a C57BL/6J BAC clone (RP23-333P11) astemplate. The STOP codon of the 5′ HR was excluded resulting in a fusionconstruct with introduced downstream sequences. The 5′ and 3′ HR PCRproducts were subcloned into pBKS-using the introduced restrictionsites, resulting in a plasmid named pBKS-Fltp-Hom Arms.

To introduce the Venus fusion reporter gene and the 3× FLAG tag into thetargeting construct the Venus sequence was amplified from pBKS-Venusvector (Nagai, Ibata et al. 2002) using primers 1126 fwdGCGGCCGCAGCCACCATGTCTAGAAT GGTGAGCAAGGGCGAGGAGCTGTTC-3′) containing anXbaI site and 1201 rev(5′-NNNACTAGTTCACTTGTCATCGTCATCCTTGTAATCGATGTCATGATCTTTATAATCACCGTCATGGTCTTTGTAGTCCTTGTACAGCTCGTCCATGCCGAGAGTGATCC-3′) containing a SpeIsite and the C-terminal 3× FLAG tag sequence. The resulting open readingframe of Venus-3× FLAG was cloned in frame with Fltp sequence into thepBKS-Fltp-HomArms vector after digestion with XbaI and SpeI.

In the next step, the PGK promoter-driven neomycin resistance geneflanked by IoxP sites (loxP-bGHpA-neo-EM7-PGK-loxP) was cloned from thePL-452 vector (Liu, Jenkins et al. 2003) via BamHI, EcoRI and subcloned3′ of the Venus-3× FLAG sequence resulting in thepBKS-Fltp-HomArms-Venus-3× FLAG-fusion-loxP-Neo-loxP. Subsequently, themini-targeting cassette was cut with NotI and HindIII and introducedinto the Fltp retrieval vector pL254 via bacterial homologousrecombination in EL350 bacteria resulting in final targeting constructpL254-Fltp-Venus-3× FLAG-fusion-loxP-bGHpA-neo-EM7-PGK-loxP. This wasconfirmed by sequencing. The targeting vector was linearized with Ascland electroporated into ES cells.

Example 11: Fltp Antibody Epitopes and Binding Specificity

Fltp antibodies were generated as described previously (Lange, Gegg etal. 2012). To analyze the Fltp protein biochemically and cellbiologically, two affinity purified polyclonal antibodies were raised inrabbit (Fltp1, Fltp116-1) against mouse Fltp using the peptide sequence:DNPDEPQSSHPSAGHT for Fltp1 and KPFDPDSQTKQKKSVTKTVQ for Fltp116-1(Pineda, Berlin, Germany). The Fltp1 epitope lies in the PRR of the lesswell conserved C terminal part of the Fltp protein (FIG. 11 B).Nevertheless, the human and murine sequences are nearly completelysimilar. The Fltp116-1 epitope lies N terminal to the Fltp1 epitope andis less conserved in human.

Additional rat monoclonal antibodies (clone #13, #28 and #43) againstthe human FLTP were prepared using the peptide sequenceKPHDPDSQKKLRKKSITKTVQ (FIG. 12). EndoC-β H1 were culture in adherence(2D culture) as described previously (Ravassard et al. (2011) and werestained as follows: cells were fixed for 10 min in 4% paraformaldehyde(PFA) at 37° C., washed 3× with PBS containing 0.2% Tween and 0.3% BSA(washing buffer) at room temperature (RT), and were permeabilized for 30min on a shaker at RT. Subsequently the cells were washed again 3× withwashing buffer at RT, and blocked for 1 h at RT in blocking buffer (PBSincluding 0.02% Tween, 10% FCS, 0.2% BSA, 3% serum). Then the primaryantibody was added in blocking solution and incubated over night at 4°C. The cells were washed 3× in washing buffer, incubated with mouseIgG2b anti Rat IgG2b antibody (dilution 1:2) for 2 h at RT. Then thecells were washed 3× in washing buffer, incubated with donkey anti-mouseIgG 488 (Invitrogen, A21202) (dilution 1:800) for 2 h at RT, washed 3×in washing buffer and embedded in Elvanol for subsequent imaging.Imaging was performed with a Leica SP5 confocal microscope according tothe manufacturer's guidelines. The 63× glycerol objective was used. FIG.12A shows that endogenous FLTP protein is localized in the cytoplasm ofEndoCβ H1 human β-cells.

For western blotting experiments (see FIG. 12B), HEK293T cellstransiently transfected with Flag-tagged FLTP Strep were used, whilenon-transfected HEK293T cells were used as controls. The human FLTPcoding sequence was obtain by PCR using cDNA from EndoC-β H1 humanβ-cells. The following primers were used:

Forward (NotI) 5′-GCGGCCGCGCCACCATGGCCACTAACTACAGTGCCAAC-3′

Reverse (Eco-RI) 5′-NNNNGAATTCTAAGGATTTGGCTGGTCTTTGGGGACC-3′.

NotI and Eco-RI restriction enzyme were used to clone human FLTP codingsequence into the pCAG Strep Flag-Tag plasmid and to generate a FLTPStrep Flag-tagged plasmid. HEK 293T were transiently transfected withFLTP Strep Flag-tagged plasmid using polyethylenimine (PEI).

For Western blotting, the samples were resuspended in RIPA buffer withprotein inhibitor and incubated on ice for 20 min. The cell lysates werecentrifuged at 14000 rpm for 30 min at 4° C. and the supernatantcontaining the protein was collected. Protein concentration wasdetermined by using the Bradford Assay. For each sample, 20 μg ofprotein were mixed 1:4 with 4×SDS loading buffer with dithiothreitol(DTT) and denatured at 95° C. for 5 min. Proteins were separated ondenaturing SDS polyacrylamide gel (10%) at 125V for approximately 1.5 h.The protein was transferred to nitrocellulose membrane by Semi-dry Blotfor 30 min at 0.44 mA and 25V. Afterwards, the membrane was incubated inPonceau-S solution to confirm the successful transfer and washed inPBS-T (PBS+0.2% Tween) to remove the color. The membrane was blockedwith 5% milk powder in PBS-T for 2 h. Subsequently, the membrane wasincubated with the primary antibody in PBS-T overnight at 4° C. whilerolling. After washing the membrane 3× for 10 min with PBS-T, thesecondary antibody in PBS-T was added and incubated for 1 h at RT. Themembrane was washed again 3× with PBS-T while shaking. The ECL (enhancedchemiluminescent solution) was prepared by mixing solution 1 and 2 at aratio of 1:1. The membrane was rinsed with the solution before wrappingit into plastic foil and placing it into an X-ray film cassette. Themembrane was exposes to a film for 10s-5 min before the film wasdeveloped.

Example 12: FLTP mRNA Expression in EndoC-β H1 Human 3-Cells

To investigate FLTP mRNA expression levels in human β-cells culturedunder 3D and 2D conditions, EndoC-β H1 human β-cells were cultured inMatrigel (3D) and compared to 2D conditions. EndoC-β H1 human β-cellline was culture in adherence (2D culture) as described previously(Ravassard et al. 2011). For 3D matrigel based cultures, EndoC-β H1 werecultured in Matrigel Matrix Growth Factor Reduced (BD Bioscience,Germany) diluted 1:2 in their respective medium.

For mRNA isolation, the miRNeasy Micro Kit (Qiagen) was used. First, 700μl QIAzol Lysis Reagent was added to the cell pellet of up to 1 millioncells. The sample was disrupted by pipetting and vortexing and incubatedfor 5 min at RT. 140 μl Chloroform was added to the sample and thenvortexed for 15s and incubated for 3 min at RT. After 15 min ofcentrifuging at 12,000×g at 4° C., the upper aqueous phase was collectedin a new tube. 100% Ethanol was added to 1.5 times volume of the sampleand mixed by pipetting. 700 μl of the sample was transferred to theRNeasy Min Elute spin column in a 2 ml collection tube and centrifugedat 8000×g for 15 s. The flow through was discarded. The DNase digest wasperformed by mixing 10 μl DNaseI and 70 μl RDD buffer and pipetting onthe membrane of the column and incubated for 15 min at RT. 700 μl BufferRWT was added to the column and centrifuged for another 15s at 8000×g.The flow-through was discarded, 500 μl RPE Buffer was added and thecolumn was centrifuged for 15 s at 8000×g. After discarding theflow-through, 500 μl of 80% ethanol was added and the column wascentrifuged for 2 min at 8000×g. The flow-through and the collectiontube were discarded again and the column was placed in another tube. Fordrying the membrane, the column was centrifuged at full speed for 5 minwith open lid. Flow-through and collection tube were discarded and thecolumn was placed in a new collection tube. The RNA was eluted byplacing 14 μl RNase-free water in the center of the membrane andcentrifuged at full speed for 1 min to collect the RNA. Purity andconcentration of isolated RNA was determined by using nanodrop. For eachsample, 1 μg of RNA was synthesized into cDNA by using the Super ScriptKit (Invitrogen, Germany). 4 μl of 5× Vilo Reaction Mix and 2 μl ofSuper Skript Enzyme Mix was added to the RNA and filled up to 20 μl withnuclease-free water. The mixture in the Eppendorf tube was placed into aheat block with the following program: 25° C. 10 min, 42° C. 60 min 85°C. 5 min.

TaqMan qPCR was assessed according with the manufacture instruction andthe following probes were used GAPdh Hs-02758991_g1 and (Fltp) C1orf192Hs01595277_g1.

As is shown in FIG. 13, FLTP mRNA expression in EndoC-β H1 human β-cellsis increased when these cells form mini-islets and cellular connections.

Example 13: The Expression of FLTP Correlates with the Expression ofOther Maturation Markers and is Induced by Compaction and Addition ofthe Noncanonical Wnt Ligand Wnt5a

To confirm that FLTP is a novel maturation marker for β cells, FLTPexpression was correlated with the expression of another maturationmarker, namely the maturation marker NKX6.1 in EndoC-β Hβ cells(described e.g. in Ravassard et al. 2011), as well as in re-aggregatedpostnatal islets (Ucn3).

EndoC-β H1 human β-cell line were cultured in adherence (2D culture) asdescribed previously (Ravassard et al. (2011)). For 3D matrigel basedcultures EndoC-β H1 were cultured in Matrigel Matrix Growth FactorReduced (BD Bioscience, Germany) diluted 1:2 in their respective medium.For analysis of WNT5a induced β-cell maturation, samples were stimulatedwith 400 ng/ml of WNT5a (R&D systems, Germany) for 12 h or 3 days.

Staining of EndoC-β Hβ cells was performed as follows: cells were fixedfor 10 min in 4% PFA at 37° C., washed 3× with PBS containing 0.2% Tweenand 0.3% BSA (washing buffer) at RT, and permeabilized for 30 min on ashaker at RT. Subsequently the cells were washed again 3× with washingbuffer at RT, blocked for 1 h at RT in blocking buffer (PBS including0.02% Tween, 10% FCS, 0.2% BSA, 3% serum). Then the primary antibody wasadded in blocking solution and incubated over night at 4° C. The cellswere washed 3× in washing buffer, incubated with the secondary antibodyfor 2 h at RT, washed 3× in washing buffer and embedded in Elvanol forsubsequent imaging. Imaging was performed with a Leica SP5 confocalmicroscope according to the manufacturer's guidelines. The 63× glycerolobjective was used.

Murine pancreatic islets of Langerhans were isolated from 5 day oldmice. After decapitation of the animals, the pancreas was dissected andsupplemented with collagenase P (1 mg/ml) in HBSS (Hanks balanced saltsolution) including 10% BSA (G-solution) and incubated for 15 min at 37°C. The pancreas was washed 2× in G-solution at RT and the islets werepicked under the stereomicroscope (Leica). The islets were incubated inRPM′ 1640 plus 5% FCS (fetal calf serum) and 1% P/S (penicillin andstreptomycin). Afterwards the islets were trypsinized with 0.05% Trypsin15 at 37° C., washed twice with PBS plus 0.3% BSA and then seeded iniBidi 8 well chambers. For immunohistochemistry the protocol mentionedabove was used. Fluorescent intensity was calculated by the Leica LAS-AF(Version 2.7.3.9723) software and the graphs were plotted with Excel.

As is shown in FIGS. 14 and 15, maturation marker expression increasesupon compaction of single cells to mini-islets in EndoC-β Hβ cells (FIG.14a-d ; FIG. 15a-d ).

Taken together, these findings of a correlation of up-regulated FLTPexpression and another β cell maturation marker confirm that FLTP is anovel maturation marker for β cell maturation.

To additionally confirm that Wnt/PCP signaling is important for theinduction of β-cell maturation, EndoC-β H1 and neonatal isolated isletswere stimulated with the noncanonical Wnt ligand Wnt5a. As shown in FIG.14 e-l and FIG. 15 e-h, Wnt5a stimulation of also resulted in increasedexpression of maturation markers.

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1. A method of distinguishing mature β cells from immature progenitor βcells by assessing expression of the biomarker Flattop (Fltp).
 2. Themethod of claim 1, the method comprising: determining the presence orabsence of the biomarker Flattop (Fltp) in a β cell; wherein thepresence of Fltp in the cell indicates that the cell is a mature β celland wherein the absence of Fltp in the cell indicates that the cell isan immature progenitor β cell.
 3. The method according to claim 2,wherein the presence or absence of Fltp is determined (i) on the nucleicacid level, (ii) on the amino acid level, or (iii) a combinationthereof.
 4. The method according to claim 2, wherein the presence orabsence of Fltp is determined by immunohistochemistry, live cellimaging, Western Blot, Northern Blot, PCR, RNA in situ hybridisation ora combination thereof.
 5. A method of identifying a compound suitablefor differentiating immature progenitor β cells into mature β cells, themethod comprising: (a) contacting a cell population comprising immatureprogenitor β cells with a test compound; and (b) subsequentlydetermining the presence or expression level of the biomarker Flattop(Fltp) in the β cells comprised in the cell population; wherein thepresence of Fltp, or an increased expression level of Fltp, in the βcells comprised in the cell population after the contacting with thetest compound is indicative of a compound suitable for differentiatingimmature progenitor β cells into mature β cells.
 6. A method ofidentifying a compound suitable for preventing the de-differentiation ofmature β cells, the method comprising: (a) culturing a cell populationcomprising mature β cells in the presence of a test compound, whereinthe cells are cultured under conditions that induce thede-differentiation of said mature β cells; and (b) subsequentlydetermining the expression level of the biomarker Flattop (Fltp) in theβ cells cultured in step (a), wherein an expression level of Fltpdetermined in step (b) that is substantially identical to the expressionlevel of Fltp in the cell population comprising mature β cells prior tothe culture in step (a) is indicative of a compound suitable forpreventing the de-differentiation of mature β cells.
 7. The methodaccording to claim 5 or 6, wherein the test compound is a compound thatactivates planar cell polarity (PCP).
 8. The method according to claim7, wherein the compound that activates planar cell polarity (PCP) is anactivator of the non-canonical Wnt/PCP pathway.
 9. A method ofdifferentiating immature progenitor β cells into mature β cells, themethod comprising: inducing the expression of Fltp in a cell populationcomprising immature progenitor β cells.
 10. The method according toclaim 9, wherein the expression of Fltp in the cells is induced byculturing the cells in the presence of a compound selected from Wnt5a,Wnt11, Wnt3a, an activator of at least one of the co-receptors ROR1, 2,RYK, MUSK, PTK7, Syndecan and/or Glypican or a compound identified bythe method of claim
 5. 11. A method of preventing de-differentiating ofmature β cells, the method comprising: inducing or maintaining theexpression of Fltp in mature β cells.
 12. The method according to claim11, wherein the expression of Fltp in the cells is induced or maintainedby culturing the cells in the presence of a compound selected fromWnt5a, Wnt11, Wnt3a, an activator of at least one of the co-receptorsROR1, 2, RYK, MUSK, PTK7, Syndecan and/or Glypican or a compoundidentified by the method of claim
 6. 13. A kit for distinguishing matureβ cells from immature progenitor β cells, the kit comprising: (a) meansfor determining the presence or absence of the biomarker Flattop (Fltp),and (b) instructions how to use the kit.
 14. A pharmaceuticalcomposition for use in treating or preventing diabetes, wherein thepharmaceutical composition comprises (an) activator(s) of Fltpexpression.
 15. The pharmaceutical composition of claim 14, wherein theactivator of Fltp expression is selected from Wnt5a, Wnt11, Wnt3a, anactivator of at least one of the co-receptors ROR1,2, RYK, MUSK, PTK7,Syndecan and/or Glypican and/or a compound identified by the method ofclaim 5.